EP0268377A2 - Glutamic acid derivatives, processes and intermediates for their preparation and pharmaceutical formulations containing them - Google Patents
Glutamic acid derivatives, processes and intermediates for their preparation and pharmaceutical formulations containing them Download PDFInfo
- Publication number
- EP0268377A2 EP0268377A2 EP87309165A EP87309165A EP0268377A2 EP 0268377 A2 EP0268377 A2 EP 0268377A2 EP 87309165 A EP87309165 A EP 87309165A EP 87309165 A EP87309165 A EP 87309165A EP 0268377 A2 EP0268377 A2 EP 0268377A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- compound
- glutamyl
- formula
- gamma
- glutamic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 15
- 230000008569 process Effects 0.000 title claims description 14
- 239000000543 intermediate Substances 0.000 title abstract description 7
- 150000002306 glutamic acid derivatives Chemical class 0.000 title description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 145
- 150000003839 salts Chemical class 0.000 claims abstract description 44
- 239000001257 hydrogen Substances 0.000 claims abstract description 37
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 37
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims abstract description 25
- 150000002431 hydrogen Chemical group 0.000 claims abstract description 19
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 125000002252 acyl group Chemical group 0.000 claims abstract description 11
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims abstract description 7
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims abstract description 5
- 125000000229 (C1-C4)alkoxy group Chemical group 0.000 claims abstract description 3
- 125000004765 (C1-C4) haloalkyl group Chemical group 0.000 claims abstract 2
- 125000001475 halogen functional group Chemical group 0.000 claims abstract 2
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 27
- 229960002989 glutamic acid Drugs 0.000 claims description 18
- CPVRCGLCOADOPV-ZDUSSCGKSA-N (2s)-2-[[4-[3-(2,6-diamino-4-oxo-1h-pyrimidin-5-yl)propylamino]benzoyl]amino]pentanedioic acid Chemical compound N1C(N)=NC(=O)C(CCCNC=2C=CC(=CC=2)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=C1N CPVRCGLCOADOPV-ZDUSSCGKSA-N 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 7
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 16
- 238000011282 treatment Methods 0.000 abstract description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 87
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 85
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 60
- 239000000243 solution Substances 0.000 description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 46
- 150000002148 esters Chemical class 0.000 description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 38
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 38
- 238000006243 chemical reaction Methods 0.000 description 34
- 239000000203 mixture Substances 0.000 description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- 235000019439 ethyl acetate Nutrition 0.000 description 29
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- 229940093499 ethyl acetate Drugs 0.000 description 27
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 26
- 235000019441 ethanol Nutrition 0.000 description 26
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 24
- 238000000921 elemental analysis Methods 0.000 description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 22
- 239000000047 product Substances 0.000 description 22
- 239000007787 solid Substances 0.000 description 21
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 19
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 19
- 239000000706 filtrate Substances 0.000 description 18
- 238000005481 NMR spectroscopy Methods 0.000 description 17
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- 239000002253 acid Chemical class 0.000 description 15
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 15
- 239000006260 foam Substances 0.000 description 14
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 14
- 238000001953 recrystallisation Methods 0.000 description 14
- 239000002904 solvent Substances 0.000 description 14
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 14
- 229960000583 acetic acid Drugs 0.000 description 13
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 13
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 12
- 235000019341 magnesium sulphate Nutrition 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 238000001914 filtration Methods 0.000 description 11
- 238000011321 prophylaxis Methods 0.000 description 11
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 10
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 229920006395 saturated elastomer Polymers 0.000 description 10
- -1 2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl Chemical group 0.000 description 9
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 9
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 235000017557 sodium bicarbonate Nutrition 0.000 description 9
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 9
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 8
- 239000000538 analytical sample Substances 0.000 description 8
- 230000000118 anti-neoplastic effect Effects 0.000 description 8
- 238000001704 evaporation Methods 0.000 description 8
- 230000008020 evaporation Effects 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 8
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical class OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- 239000004480 active ingredient Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 125000004005 formimidoyl group Chemical group [H]\N=C(/[H])* 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 238000006722 reduction reaction Methods 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 208000028018 Lymphocytic leukaemia Diseases 0.000 description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 6
- 238000003818 flash chromatography Methods 0.000 description 6
- 235000019152 folic acid Nutrition 0.000 description 6
- 239000011724 folic acid Substances 0.000 description 6
- 208000003747 lymphoid leukemia Diseases 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 239000006188 syrup Substances 0.000 description 6
- 235000020357 syrup Nutrition 0.000 description 6
- PVFCXMDXBIEMQG-JTQLQIEISA-N (2s)-2-(phenylmethoxycarbonylamino)pentanedioic acid Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 PVFCXMDXBIEMQG-JTQLQIEISA-N 0.000 description 5
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical class C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 239000012267 brine Substances 0.000 description 5
- KVUAALJSMIVURS-ZEDZUCNESA-L calcium folinate Chemical compound [Ca+2].C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 KVUAALJSMIVURS-ZEDZUCNESA-L 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 125000006308 propyl amino group Chemical group 0.000 description 5
- 239000012258 stirred mixture Substances 0.000 description 5
- 239000003826 tablet Substances 0.000 description 5
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 4
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 4
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 4
- 241000202915 Spiroplasma citri Species 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 231100000263 cytotoxicity test Toxicity 0.000 description 4
- 230000020176 deacylation Effects 0.000 description 4
- 238000005947 deacylation reaction Methods 0.000 description 4
- HOXINJBQVZWYGZ-UHFFFAOYSA-N fenbutatin oxide Chemical compound C=1C=CC=CC=1C(C)(C)C[Sn](O[Sn](CC(C)(C)C=1C=CC=CC=1)(CC(C)(C)C=1C=CC=CC=1)CC(C)(C)C=1C=CC=CC=1)(CC(C)(C)C=1C=CC=CC=1)CC(C)(C)C1=CC=CC=C1 HOXINJBQVZWYGZ-UHFFFAOYSA-N 0.000 description 4
- 229960000304 folic acid Drugs 0.000 description 4
- OWQDWQKWSLFFFR-WDSKDSINSA-N gamma-Glu-Glu Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(O)=O)CCC(O)=O OWQDWQKWSLFFFR-WDSKDSINSA-N 0.000 description 4
- 235000013922 glutamic acid Nutrition 0.000 description 4
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- 230000007062 hydrolysis Effects 0.000 description 4
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- 239000002808 molecular sieve Substances 0.000 description 4
- YLSRAWUBPSMJOD-UHFFFAOYSA-N n-[6-(diacetylamino)-4-oxo-5-(2-oxoethyl)-1h-pyrimidin-2-yl]acetamide Chemical compound CC(=O)NC1=NC(=O)C(CC=O)=C(N(C(C)=O)C(C)=O)N1 YLSRAWUBPSMJOD-UHFFFAOYSA-N 0.000 description 4
- SPOPKLMNYTWYDT-UHFFFAOYSA-N n-[6-(diacetylamino)-4-oxo-5-(3-oxopropyl)-1h-pyrimidin-2-yl]acetamide Chemical compound CC(=O)NC1=NC(N(C(C)=O)C(C)=O)=C(CCC=O)C(=O)N1 SPOPKLMNYTWYDT-UHFFFAOYSA-N 0.000 description 4
- HQDMWAFMKYTDIK-UHFFFAOYSA-N n-[6-(diacetylamino)-5-(3,3-diethoxypropyl)-4-oxo-1h-pyrimidin-2-yl]acetamide Chemical compound CCOC(OCC)CCC1=C(N(C(C)=O)C(C)=O)N=C(NC(C)=O)NC1=O HQDMWAFMKYTDIK-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 0 *CC(CO)N* Chemical compound *CC(CO)N* 0.000 description 3
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 3
- SZNZPWBACCDJIR-UHFFFAOYSA-N 4-[3-(2,6-diamino-4-oxo-1h-pyrimidin-5-yl)propylamino]benzoic acid Chemical compound O=C1NC(N)=NC(N)=C1CCCNC1=CC=C(C(O)=O)C=C1 SZNZPWBACCDJIR-UHFFFAOYSA-N 0.000 description 3
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
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- WBTXVEYIWOPUFI-UHFFFAOYSA-N n-[6-(diacetylamino)-5-(2,2-diethoxyethyl)-4-oxo-1h-pyrimidin-2-yl]acetamide Chemical compound CCOC(OCC)CC1=C(N(C(C)=O)C(C)=O)NC(NC(C)=O)=NC1=O WBTXVEYIWOPUFI-UHFFFAOYSA-N 0.000 description 3
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- RKMWDDTUARZQSJ-UHFFFAOYSA-N 2,6-diamino-5-(3,3-diethoxypropyl)-1h-pyrimidin-4-one Chemical compound CCOC(OCC)CCC1=C(N)N=C(N)NC1=O RKMWDDTUARZQSJ-UHFFFAOYSA-N 0.000 description 2
- FGDVWTAEUHOXAY-UHFFFAOYSA-N 4-[3-(2,6-diamino-4-oxo-1h-pyrimidin-5-yl)propyl-(2,2,2-trifluoroacetyl)amino]benzoic acid Chemical compound O=C1NC(N)=NC(N)=C1CCCN(C(=O)C(F)(F)F)C1=CC=C(C(O)=O)C=C1 FGDVWTAEUHOXAY-UHFFFAOYSA-N 0.000 description 2
- DVNYTAVYBRSTGK-UHFFFAOYSA-N 5-aminoimidazole-4-carboxamide Chemical compound NC(=O)C=1N=CNC=1N DVNYTAVYBRSTGK-UHFFFAOYSA-N 0.000 description 2
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
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- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
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- UDGSVBYJWHOHNN-UHFFFAOYSA-N n',n'-diethylethane-1,2-diamine Chemical compound CCN(CC)CCN UDGSVBYJWHOHNN-UHFFFAOYSA-N 0.000 description 1
- MENVFLXNINZVEW-UHFFFAOYSA-N n-[6-(diacetylamino)-5-(1,1-diethoxyethyl)-4-oxo-1h-pyrimidin-2-yl]acetamide Chemical compound CCOC(C)(OCC)C1=C(N(C(C)=O)C(C)=O)NC(NC(C)=O)=NC1=O MENVFLXNINZVEW-UHFFFAOYSA-N 0.000 description 1
- BPDNTMMYJVGANK-UHFFFAOYSA-N n-[6-oxo-5-(3-oxopropyl)-1h-pyrimidin-2-yl]acetamide Chemical compound CC(=O)NC1=NC=C(CCC=O)C(=O)N1 BPDNTMMYJVGANK-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- HQOJMTATBXYHNR-UHFFFAOYSA-M thallium(I) acetate Chemical compound [Tl+].CC([O-])=O HQOJMTATBXYHNR-UHFFFAOYSA-M 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 125000005424 tosyloxy group Chemical group S(=O)(=O)(C1=CC=C(C)C=C1)O* 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229940051021 yellow-fever virus Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/48—Two nitrogen atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a class of novel glutamic acids, acid esters and salts, processes and intermediates for their preparation, pharmaceutical formulations containing them, and to their use in human and veterinary medicine and agriculture.
- Folic acid or more particularly its coenzyme form, tetrahydrofolic acid or a related derivative, is required by organisms for the biosynthesis of the nucleotide, adenylic acid, guanylic acid and thymidylic acid. Without this essential vitamin, organisms cannot grow and may eventually die. Subsequent to the elucidation of the structure of folic acid, a large number of potential antagonists has been synthesised as candidate chemotherapeutic agents. The majority of these antagonists inhibit dihydrofolate reductase, which is responsible for the conversion of dihydrofolic acid into tetrahydrofolic acid.
- Examples of such antagonists include a number of pyrimidyl analogues of folic acid, such as N-[2-(2-amino-4-hydroxy-6-methyl-5-pyrimidyl)-3-propyl]- p -aminobenzoyl-L-glutamic acid ( J . Pharm . Sci ., 1963, 52 , 840 to 843), and the corresponding 6-phenyl compound which, compared to the 6-methyl compound, is an even more effective inhibitor of dihydrofolate reductase ( J . Med . Chem ., 1963, 6 , 664 to 669).
- tetrahydrohomofolic acid which is an analogue of tetrahydrofolic acid differing only in that it has an additional methylene group between the C-9 and N-10 atoms of the folate molecule.
- This antagonist is effective against bacteria ( J . Am . Chem . Soc ., 1964, 86 , 308 to 309) and tumour cell growth ( Cancer Res ., 1966, 26 , 2374 to 2379).
- a structurally distinct class of novel substituted glutamic acid and acid esters has now been discovered in which the glutamic acid or acid ester is substituted by a pyrimidylalkylaminobenzoyl chain characterised in that the pyrimidyl moiety is 2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl. They have, moreover, been found to possess anti-neoplastic activity in that the compounds are able to inhibit the unregulated multiplication and proliferation of undifferentiated cells. Such activity has been demonstrated against lymphocytic and connective tissue cells in the lymophocytic leukemia P388/0 and the cell culture cytotoxicity tests, both of which are described hereinafter.
- the present invention provides a compound of formula (I): wherein R1 is hydrogen, C 1-4 alkyl, acetyl or formyl; R2 and R3 are the same or different and are hydrogen or C 1-4 alkyl; R4 is NR11R12; R11, R12, R5 and R6 are the same or different and are hydrogen, C 1-4 alkyl or C 1-12 acyl; R7, R8, R9 and R10 are the same or different and are hydrogen, halo, C 1-4 haloakyl, C 1-4 alkyl and C 1-4 alkoxy; and n is 2, 3, 4 or 5; m is o or an integar from 1 to 6; or a salt thereof.
- R1 is hydrogen, C 1-4 alkyl, acetyl or formyl
- R2 and R3 are the same or different and are hydrogen or C 1-4 alkyl
- R4 is NR11R12
- R11, R12, R5 and R6 are the same or different and are
- R1 when C 1-4 alkyl, include methyl. It is, however, preferred that R1 is hydrogen or formyl.
- R2 and R3 when C 1-4 alkyl, include methyl and ethyl. It is, however, preferred that R2 and R3 are both hydrogen.
- n is 3.
- m is 0 to 2.
- m is 0
- the compounds of the present invention have an asymmetric carbon atom indicated by the asterisk in formula (I) and are, therefore, capable of existing as optical isomers. Although all such isomers, individually and as mixtures, are included within the scope of the present invention, the L -optical isomers are preferred.
- salts of the compounds of the present invention there are included acid addition salts derived from either of the two terminal amino groups that substitute the pyrimidyl moiety or from the amino group present in the chain between the phenylene and the -(CH2) n - moieties and salts comprising an anionic species derived from a compound of formula (I), wherein one or both of R2 and R3 is or are hydrogen, and a cation.
- the anti-neoplastic activity resides in the moiety derived from the compound of the invention as defined herein and the identity of the other component is of less importance although for therapeutic and prophylactic purposes it is, preferably, pharmaceutically acceptable to the patient.
- Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic and arylsulphonic, for example p -toluenesulphonic, acids.
- mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
- organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic and arylsulphonic, for example p -toluenesulphonic, acids.
- salts comprising an anionic species derived from a compound of formula (I), wherein one or both of R2 and R3 is or are hydrogen, and a cation include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth salts, such as magnesium and calcium salts, and salts formed with organic bases, for example, amino salts derived from mono-, di, or tri-(lower alkyl) or (lower alkanol)amines, such as triethanolamine and diethylaminoethylamine, and salts with heterocyclic amines such as piperidine, pyridine, piperazine and morpholine.
- the pharmaceutically acceptable salts together with the salts which are not thus acceptable have utility in the isolation and/or the purification of the compounds of the invention, and the unacceptable salts are also useful in being convertible to the acceptable salts by techniques well known in the art.
- the present invention also provides a process for the preparation of a compound of formula (I), as defined herein, or a salt thereof, which comprises deacylating a compound of formula (II): wherein R1 is hydrogen or C 1-4 alkyl; R2, R3, R7, R8, R9 and R10 and n and m are as defined in Claim 1; one of the groups R5, R6, R11 and R12 being C 1-12 acyl, the other groups R5, R6, R11 and R12 being the same or different and are hydrogen or C 1-12 acyl, and optionally thereafter (i) in the case where, in the resulting compound of formula (I) R1 is hydrogen, formylating the compound so as to prepare the corresponding compound of formula (I), wherein R1 is formyl; and/or (ii) converting R2, R3, R5, R6, R11 and R12 in the resulting compound of formula (I) into another R2, R3, R5, R6, R11 and R12; and optionally forming a salt.
- R1
- R5, R6, R11 and R12 when C 1-12 acyl are carboxylic C 1-12 acyl.
- R5, R6, R11 and R12, when C 1-12 acyl are the same or different and are C 1-12 carboxylic acyl, in particular C 1-6 alkanoyl, especially acetyl.
- the deacylation of a compound of formula (II) may be carried out conventionally.
- the deacylation is, preferably, carried out at an elevated temperature in an alcoholic solvent, such as ethanol or ethanol containing a small amount of 2-mercaptoethanol so as to prevent any oxidation of the resulting compound of formula (I), in the presence of a base, such as sodium hydroxide.
- an alcoholic solvent such as ethanol or ethanol containing a small amount of 2-mercaptoethanol
- the product of the deacylation is a compound of formula (I), wherein R2 and R3 are both hydrogen.
- the resulting compound of formula (I), wherein R2 and R3 are both hydrogen is esterified as described hereinafter.
- the optional formylation of the resulting compound of formula (I), wherein R1 is hydrogen, is, preferably, carried out by reacting the compound with acetic formic anhydride at an elevated temperature.
- Examples of the optional conversion of R2 and/or R3 in the resulting compound of formula (I) into another R2 and/or R3 include the optional conversion of hydrogen into C 1-4 alkyl using conventional esterification reagents and conditions, for example a C 1-4 alkanol in the presence of an acid. This may be a useful optional conversion to carry out in the event that, in the preparation of a compound of formula (I), wherein one or both of R2 and R3 is or are C 1-4 alkyl, the compound undergoes deesterification as described hereinbefore. In such circumstances, the desired ester may be prepared from the free acid as just described.
- the compounds of formula (II) may be prepared by reducing a mixture of a compound of formula (III): where R4 to R6 and n are as defined herein, and a compound of formula (VI): wherein R1, R2, R3, R7, R8, R9 and R10 are as defined herein; and, in the case where, in the resulting compound of formula (II) R1 is hydrogen, optionally alkylating the compound so as to prepare the corresponding compound of formula (II), wherein R1 is C 1-4 alkyl.
- the reduction of a mixture of the compounds of formulae (III) and (VI) may be carried out in accordance with conventional reductive alkylation reactions. It is however, preferred that the reduction is effected with sodium cyanoborohydride in a solvent in the presence of an acid, such as acetic acid which may also function as the solvent.
- the optional alkylation of the resulting compound of formula (II), wherein R1 is hydrogen is, preferably, carried out by reductive alkylation in which a mixture of the compound of formula (II) and a C 1-4 aldehyde are reduced with sodium cyanoborohydride in the presence of an acid, such as acetic acid.
- the compounds of formula (II), wherein n is 3, may also, although less preferably, be prepared by reducing a mixture of a compound of formula (IV): wherein R4 and R5 are as defined herein, and a compound of formula (VI) as defined herein; and, in the case where, in the resulting compound of formula (II) where R1 is hydrogen, optionally alkylating the compound so as to prepare the corresponding compound of formula (II), wherein R1 is C 1-4 alkyl.
- the reduction of a mixture of the compounds of formulae (IV) and (VI) may be carried out analogously to the reduction of a mixture of the compounds of formulae (III) and (VI) in which sodium cyanoborohydride is used in the presence of an acid, such as acetic acid, to effect the reduction. It is, however, preferred, in the reduction of the mixture of the compounds of formulae (VI) and (IV), that acetic acid is not used also as the solvent but that, for example, methanol or a mixture of methanol and 2-methoxyethanol is used in this regard. It is also preferred that the reduction is carried out in the presence of a dehydrating agent, such as molecular sieves.
- a dehydrating agent such as molecular sieves.
- the compounds of formulae (III) and (IV) may both be prepared by hydrolysis of a compound of formula (V): wherein R11, R5, R6 and n are as defined herein and R13 is C 1-4 alkyl, such as ethyl.
- the hydrolysis may be carried out in a solvent, such as dichloromethane or acetone, in the presence of an acid, such as oxalic acid or 4-toluenesulphonic acid.
- a solvent such as dichloromethane or acetone
- an acid such as oxalic acid or 4-toluenesulphonic acid.
- the hydrolysis may also be carried out simply in water at room or a slightly elevated temperature.
- the compound of formula (V) may be prepared by acylating a compound of formula (VII): wherein R13 and n are as defined herein.
- acylation of a compound of formula (VII) may be carried out conventionally using, for example, an acid anhydride in the presence of a base, such as pyridine.
- the compound of formula (VII) may be prepared by reacting a compound of formula (VIII): wherein R13 and n are as defined herein and R14 is C 1-4 alkyl, such as ethyl, and guanidine.
- reaction between the compound of formula (VIII) and guanidine may be carried out conventionally, for example, in a solvent, such as ethanol, in the presence of a base, such as sodium methoxide, at an elevated temperature.
- a solvent such as ethanol
- a base such as sodium methoxide
- the compound of formula (VIII) may be prepared by reacting a compound of formula (IX): wherein R14 is as defined herein, and a compound of formula (X): L-(CH2) n-1 CH(OR13)2 (X) wherein R13 and n are as defined herein and L is a leaving group, such as chloro, bromo, iodo, or tosyloxy.
- reaction between the compounds of formulae (IX) and (X) may be carried out conventionally, for example, in a solvent, such as ethanol, in the presence of a base, such as sodium methoxide.
- a solvent such as ethanol
- a base such as sodium methoxide
- the compounds of formulae (VI), (IX) and (X) are commercially available, or may be obtained by carrying out a published process for their preparation, or by carrying out a process analogous to a published process for the preparation of structurally analogous compounds.
- the compounds of formula (VI) may be obtained by using the process described in J . Am . Chem . Soc ., 1958, 80 , page 5778 et seq .
- the compounds of formulae (II), (III), (IV) and (V), on the other hand, are novel intermediates of use in the preparation of the compounds of formula (I) and, thus, represent part of the present invention.
- the present invention further provides a pharmaceutical formulation, for human or veterinary application, which comprises a compound of the present invention or a pharmaceutically acceptable salt thereof, as hereinbefore defined, and a pharmaceutically acceptable carrier thereof.
- the pharmaceutical formulation may optionally contain other therapeutic agents that may usefully be employed in conjunction with the compound or salt of the present invention, for example a dihydrofolate reductase inhibitor that is capable of enhancing the antineoplastic activity of the compounds and salts of the present invention.
- a dihydrofolate reductase inhibitor that is capable of enhancing the antineoplastic activity of the compounds and salts of the present invention.
- pharmaceutically acceptable as used herein in relation to the carrier is used in the sense of being compatible with the compound or salt of the invention employed in the formulation and with any other therapeutic agent that may be present, and not being detrimental to the recipient thererof.
- the carrier itself may constitute one or more excipients conventionally used in the art of pharmacy that enable the compound or salt of the present invention, and any other therapeutic agent that may be present, to be formulated as a pharmaceutical formulation.
- the pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration although the most suitable route will probably depend upon, for example, the condition and identity of the recipient.
- the formulations may conventionally be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e. the compound or salt of the present invention, with the carrier.
- the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation.
- the pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units, such as a capsule, cachet, tablet, or lozenge, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid such as a syrup, elixir or a draught, or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
- the formulation may also be a bolus, electuary or paste.
- a tablet is the most convenient pharmaceutical formulation suitable for oral administration.
- a tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier.
- Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating agent and/or a surface active agent.
- Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
- the tables may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient.
- the pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain, for example, a anti-oxidant, a buffer, a bacteriostat and a solute which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent.
- the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- compositions of the present invention suitable for rectal administration may be presented as a suppository containing, for example, cocoa butter and polyethylene glycol.
- the compounds and salts of the present invention have anti-neoplastic activity as demonstrated hereinafter in the lymphocytic leukemia P388/0 and the cell culture cytotoxicity tests, in which a representative number of the compounds of the present invention is shown to be active against lymphocytic and connective tissue cell lines. It has thus been established that the compounds of the present invention are able to inhibit neoplastic growth. Therefore, the compounds and salts of the present invention are of use in human and veterinary medicine and in particular in the treatment of prophylaxis of neoplastic growth, especially lymphocytic leukemia and malignant tumours (otherwise known generally as cancers).
- the present invention yet further provides a method for the treatment or prophylaxis of neoplastic growth in an animal, which comprises administering to the animal a therapeutically or prophylactically effective amount of a compound or salt of the present invention.
- a compound or salt of the present invention for use in human or veterinary medicine and in particular for use in the treatment or prophylaxis of a neoplastic growth.
- the animal requiring treatment or prophylaxis with a compound or salt of the present invention is usually a human or non-human mammal.
- neoplastic growth requiring treatment or prophylaxis include lymphocytic leukemia and malignant tumours.
- the antineoplastic activity of the compounds and salts of the present invention may be enhanced by a dihydrofolate reductase inhibitor, for example 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3- d ]pyrimidine hydrochloride. Therefore, it may be advantageous to employ with the compounds and salts of the present invention a dihyrofolate reductase inhibitor in the treatment or prophylaxis of neoplastic growth.
- a dihydrofolate reductase inhibitor for example 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3- d ]pyrimidine hydrochloride. Therefore, it may be advantageous to employ with the compounds and salts of the present invention a dihyrofolate reductase inhibitor in the treatment or prophylaxis of neoplastic growth.
- the route by which the compound or salt of the present invention is administered to the animal may be oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous or rectal). If the compound or salt is presented in the form of a pharmaceutical formulation, which, as mentioned hereinbefore, is preferred, then the actual formulation employed will of course depend on the route of administration elected by the physician or veterinarian. For example, if oral administration is preferred, then the pharmaceutical formulation employed is, preferably, one which is suitable for such a route.
- a therapeutically or prophylactically effective amount of a compound or salt of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment or prophylaxis and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian.
- an effective amount of a compound of the present invention for the treatment or prophylaxis of neoplastic growth, in particular lymphocytic leukemia or a malignant tumour will generally be in the range of 0.5 to 600 mg/kg body weight per day.
- the actual amount per day would usually be from 490 to 14,000mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same.
- An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se .
- the treatment or prophylaxis of neoplastic growth with a compound of the present invention may at times require the administration to the animal of an antidote or rescue agent.
- agents include leucovorin, hypoxanthine and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), although leucovorin is the most preferred.
- the compounds of Examples 1, 2 and 3 hereinafter have also been found to have activity against the mycoplasma, Spiroplasma citri . These compounds are, therefore, useful in the treatment or prophylaxis of citrus plants that are infected with this microorganism.
- the compounds may be applied to the plants by methods known in the art, such as spraying, dusting, incorporating into the soil or injecting into the plant.
- the compound of Example 1 has also been found to possess activity against certain bacterial organisms that are unable to synthesize their own folic acid and that, therefore, require preformed folate. These organisms with the Lactobacillus casei and Streptococcus faecium . And together with the compound of Example 3, the compound of Example 1 has further been found to possess some activity against yellow fever virus.
- Method 1 A solution of 3-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propionaldehyde diethyl acetal (1.00 g, 2.6 mmol) in distilled water (30 ml) was stirred at room temperature for 18 hours. This solution was extracted with ethyl ether (3 x 50 ml), and chloroform (5 x 50 ml). The chloroform extracts were combined, washed with water (25 ml), brine (25 ml), dried (MgSO4) and spin evaporated in vacuo .
- This foam was added to a column (40mm x 180 mm) of Silica Gel 60 wetted with ethyl acetate and eluted using the "flash chromatography technique" (W.C. Still, M. Kahn, and A Mitra, J . Org . Chem ., 43 , 2923 1987).
- the column was washed with 1 litre of ethyl acetate followed by methanol/dichloromethane (1:9) to elute the product.
- the fractions containing product were combined and spin evaporated in vacuo to yield 8.2 g of the acetylated ester intermediate, mp 175-180°C.
- Method 1 slurry of Silica Gel 60 (6.0 g, Merck ⁇ 7734, 70-230 mesh) and aqueous oxalic acid (10%) (0.6 ml) in dichloromethane (12 ml) was stirred for 15 minutes. 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-acetaldehyde diethyl acetal (1.0 g, 2.7 mmol) was added and the mixture was stirred for 18 hours. Sodium bicarbonate (0.2 g, 2.4 mmol) was added and after 10 minutes the slurry was filtered and washed with ethyl acetate.
- the 20 hr reaction was done in an amber, gas-tight bottle in a gently shaking 37°C H2O bath.
- the argon-saturated 20 ml reaction contained 4 #moles 14C- 1 labeled in the benzoyl carbonyl, 40 ⁇ moles L-glutamic acid, 1 mmole KCl, 4mg bovine serum albumin, 1.4 mmoles 2-mercaptoethanol (2-MET), 0.1 mmole adenosine-5 ⁇ -triphosphate, 0.2 mmole MgCl2, 2 mmole Tris-HCl pH 8.6 (37°) and 50 units of enzyme.
- HPLC analysis of 100 ⁇ l of the reaction mixture after 20 hr revealed two 14C-containing products and no remaining 14C ( 1 ).
- the reaction mixture was adjusted to pH 1 with 2.8 ml of 10% wt-vol trifluoroacetic acid (TFA). It was protected from light during all handling. A 50 ml portion was desalted on a Waters C18 Sep-Pak which had been prewashed with 5 ml of 2-propanol (ISP), 10 ml H2O and 5 ml 0.1% wt/vol TFA. The 14C- containing products were retained on the Sep-Pak during a 10 ml 0.1% wt/vol TFA wash and eluted with 8 ml 0.1% TFA, 50% ISP 50 #1 2-MET. The eluant was argon saturated in a sealed amber bottle, shell-frozen and lyophilized.
- ISP 2-propanol
- eluant was argon saturated in a sealed amber bottle, shell-frozen and lyophilized.
- the dried sample was dissolved in 1.0 ml 3% acetonitrile (ACN) in HPLC solvent (0.14 M sodium acetate pH 6.4, 0.05% triethylamine) and passed through a 0.45 ⁇ m Millipore HV filter.
- the filtrate (840 ⁇ l) was chromatographed on a 10 cm Waters C18 Nova-Pak Radial Compression Column at 1 ml/min (1.5% ACN in HPLC solvent for 15 minutes followed by 2.4% ACN in HPLC solvent). Two 14C-containing peaks, identified as 3 and 2 were eluted at 27 and 33 minutes, respectively.
- the glutamate chain length of 3 and 2 was established after cleavage of the compounds at the propylamino C-N bond. (A 1.0 ml solution at pH 9 was oxidized with 100 ⁇ l 2% KMnO4. After one minute, 80 ⁇ l 30% H2O2 was added to precipitate KMnO2. Excess H2O2 was destroyed by the addition of 20 ⁇ l of 11 ⁇ g/ml beef liver catalase, Worthington Biochemical Corp).
- the filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 40 ml), 5% aqueous citric acid (2 x 40 ml) and saturated brine (2 x 40 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo .
- the product was purified by flash chromotography on silica gel 60 (150 g, 230- 400 mesh, EM Reagents) with hexanes: ethyl acetate (1:1).
- the reaction was stirred for 16 hours and filtered.
- the filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2x50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 50 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo .
- the product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with ethyl acetate : hexanes (1:1).
- the reaction was stirred for 17 hours and filtered.
- the filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 50 ml), dried with magnesium sulfate, filtered and spin evaporated in vacuo .
- the product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with hexanes: ethyl acetate (3:7).
- the reaction was stirred for 19 hours and filtered.
- the filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 25 ml), 5% aqueous citric acid (2 x 25 ml) and saturated brine (2 x 25 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo.
- the product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents)with hexanes:ethyl acetate (1:2).
- the reaction was stirred for 18 hours and filtered.
- the filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 25 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo .
- the product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with hexanes:ethyl acetate (1:3).
- the product was separated from a mixture by flash chromatography on silica gel 60 (100 g; 230-400 mesh, EM Reagents) with ethyl acetate. Appropriate fractions were combined on the basis of TLC correlation, and solvent was removed by spin evaporation in vacuo .
- the resulting yellow solution was spin evaporated in vacuo to a beige solid, which was subsequently dissolved in 5.0 ml of water.
- the aqueous solution was injected into a column of C-18 (comprised of a series of five Rainin Spice cartridges connected in series preequillibrated with water. The column was washed with 50 ml of water before the product was eluted with 20% acetonitrile. Solvent was removed by spin evaporation and freeze-drying. The residue was partitioned between a 1:1 mixture of ethylacetate and water.
- the tests employed for the evaluation of anti-neoplastic activity of the compounds of the present invention are essentially those used in the Tumor Panel of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, A. Goldin, et al., Methods in Cancer Research, Vol. XVI, p. 165, Academic Press (1979). Some modifications, in dose level and schedule have been made to increase the testing efficiency.
- CD2-F1-mice of the same sex, weighing withi a 3 g range surrounding 20 g, are used for this test.
- Control and test animals are injected intraperitoneally with a suspension of 106 viable P388/0 tumor cells on day 0.
- each dose level group contains six animals.
- the test compound is prepared either in physiologic saline containing 0.05% Tween 80 or distilled water containing 5% dextrose and is administered intraperitoneally on the indicated schedule relative to tumor implant. Doses are on a mg/kg basis according to individual animals' body weights. The day of death for each animal is recorded, the median identified for each group and the percent increase in life span (% ILS) is calculated from the ratio of survival time of treated to control groups. The criterion for activity is % ILS greater than or equal to 20%.
- the compound of the invention included in this test was the compound of Example 1, namely N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-propylamino]benzoyl]- L -glutamic acid and the results are as follows.
- the two routine indicator cell lines used are 1.) D98, a twice- cloned derivative of Detroit 98s from American Type Culture Collection (ATCC) strain CCL 18.1, from human sternal bone marrow and 2.) L, a cloned derivative from NCTC 929 (ATCC CCL1,) a C3H/An from mouse connective tissue. Inoculation densities for L and D98 cells are 1.0 and 1.5 x 105 cells, per cm2, respectively. Control cells are grown in Eagle's MEM(Earle's salts, 10% horse serum, 100 units/ml potassium penicillin G and 100 ⁇ g/ml streptomycin) and should show at least two doublings during the test period.
- D98 a twice- cloned derivative of Detroit 98s from American Type Culture Collection (ATCC) strain CCL 18.1, from human sternal bone marrow
- L a cloned derivative from NCTC 929 (ATCC CCL1,) a C3H/An from mouse connective
- Test plates are seeded with similar quantities of cells and are grown in the same medium with test compounds added. Total "cell counts are made with an electronic cell counter after 70-76 hours of incubation with one medium change at 20-26 hours. Dose response curves are prepared by plotting percent of control vs. concentration of compound.
- the compounds of the present invention are active in both the lymphocytic leukemia P388/0 and the cell culture cytotoxicity tests and, therefore, that the compounds have anti-neoplastic activity and are able to inhibit neoplastic growth.
- Frozen broth cultures are diluted to a predetermined titre of 100 color changing units for the test.
- Organisms are grown in Difco PPLO broth base with added serum, glucose, phenol red, ampicillin, thallium acetate and yeast extract. Incubation is at 32oC. Incubation with varying levels of the test compound in the above medium are performed in well plates and an amount of Spiroplasma citri cells is added that will produce a color change by a fixed time of growth. The lowest concentration of compound that fails to produce a color change or gives a two day delay in color change indicates minimum inhibitory concentration.
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Abstract
Description
- The present invention relates to a class of novel glutamic acids, acid esters and salts, processes and intermediates for their preparation, pharmaceutical formulations containing them, and to their use in human and veterinary medicine and agriculture.
- Folic acid, or more particularly its coenzyme form, tetrahydrofolic acid or a related derivative, is required by organisms for the biosynthesis of the nucleotide, adenylic acid, guanylic acid and thymidylic acid. Without this essential vitamin, organisms cannot grow and may eventually die. Subsequent to the elucidation of the structure of folic acid, a large number of potential antagonists has been synthesised as candidate chemotherapeutic agents. The majority of these antagonists inhibit dihydrofolate reductase, which is responsible for the conversion of dihydrofolic acid into tetrahydrofolic acid. Examples of such antagonists include a number of pyrimidyl analogues of folic acid, such as N-[2-(2-amino-4-hydroxy-6-methyl-5-pyrimidyl)-3-propyl]-p-aminobenzoyl-L-glutamic acid (J. Pharm. Sci., 1963, 52, 840 to 843), and the corresponding 6-phenyl compound which, compared to the 6-methyl compound, is an even more effective inhibitor of dihydrofolate reductase (J. Med. Chem., 1963, 6, 664 to 669).
- One folic acid antagonist that does not appear to inhibit dihydrofolate reductase is tetrahydrohomofolic acid which is an analogue of tetrahydrofolic acid differing only in that it has an additional methylene group between the C-9 and N-10 atoms of the folate molecule. This antagonist is effective against bacteria (J. Am. Chem. Soc., 1964, 86, 308 to 309) and tumour cell growth (Cancer Res., 1966, 26, 2374 to 2379). It has been reported that, at least in relation to the effectiveness of tetrahydrohomofolic acid against mouse Sarcoma 180 cells, the active agent is actually a metabolite which blocks the biosynthesis of N-formylglycinamide ribonucleotide (Mol. Pharmacol., 1975, 11, 319 to 325). The structural identity of this metabolite has not yet been fully elucidated although one possibility put forward is that it is 5,11-methenyltetrahydro homofolic acid (J. Med. Chem., 1981, 24, 1086 to 1088). A proposal for the mechanism of action of this compound has also been put forward (Mol. Pharmacol., 1983, 25, 294 to 302) but it awaits confirmation.
- A structurally distinct class of novel substituted glutamic acid and acid esters has now been discovered in which the glutamic acid or acid ester is substituted by a pyrimidylalkylaminobenzoyl chain characterised in that the pyrimidyl moiety is 2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl. They have, moreover, been found to possess anti-neoplastic activity in that the compounds are able to inhibit the unregulated multiplication and proliferation of undifferentiated cells. Such activity has been demonstrated against lymphocytic and connective tissue cells in the lymophocytic leukemia P388/0 and the cell culture cytotoxicity tests, both of which are described hereinafter.
- Accordingly, the present invention provides a compound of formula (I):
- Examples of R¹, when C1-4 alkyl, include methyl. It is, however, preferred that R¹ is hydrogen or formyl.
- Examples of R² and R³, when C1-4 alkyl, include methyl and ethyl. It is, however, preferred that R² and R³ are both hydrogen.
- Preferably, n is 3.
Suitably m is 0 to 2.
Preferably, m is 0 - The compounds of the present invention have an asymmetric carbon atom indicated by the asterisk in formula (I) and are, therefore, capable of existing as optical isomers. Although all such isomers, individually and as mixtures, are included within the scope of the present invention, the L-optical isomers are preferred.
- Of the compounds exemplified hereinafter, those that are preferred include the compound of Example 1.
- As salts of the compounds of the present invention, there are included acid addition salts derived from either of the two terminal amino groups that substitute the pyrimidyl moiety or from the amino group present in the chain between the phenylene and the -(CH₂)n- moieties and salts comprising an anionic species derived from a compound of formula (I), wherein one or both of R² and R³ is or are hydrogen, and a cation. In both types of salts, the anti-neoplastic activity resides in the moiety derived from the compound of the invention as defined herein and the identity of the other component is of less importance although for therapeutic and prophylactic purposes it is, preferably, pharmaceutically acceptable to the patient. Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic and arylsulphonic, for example p-toluenesulphonic, acids. Examples of salts comprising an anionic species derived from a compound of formula (I), wherein one or both of R² and R³ is or are hydrogen, and a cation include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth salts, such as magnesium and calcium salts, and salts formed with organic bases, for example, amino salts derived from mono-, di, or tri-(lower alkyl) or (lower alkanol)amines, such as triethanolamine and diethylaminoethylamine, and salts with heterocyclic amines such as piperidine, pyridine, piperazine and morpholine. The pharmaceutically acceptable salts together with the salts which are not thus acceptable have utility in the isolation and/or the purification of the compounds of the invention, and the unacceptable salts are also useful in being convertible to the acceptable salts by techniques well known in the art.
- The present invention also provides a process for the preparation of a compound of formula (I), as defined herein, or a salt thereof, which comprises deacylating a compound of formula (II):
- Preferably, R⁵, R⁶, R¹¹ and R¹² when C1-12 acyl, are carboxylic C1-12 acyl. Most preferably, R⁵, R⁶, R¹¹ and R¹², when C1-12 acyl, are the same or different and are C1-12 carboxylic acyl, in particular C1-6 alkanoyl, especially acetyl.
- The deacylation of a compound of formula (II) may be carried out conventionally. In the preferred case where R⁵, R⁶, R¹¹ and R¹², when C1-12 acyl, are the same or different and are C1-6 alkanoyl, the deacylation is, preferably, carried out at an elevated temperature in an alcoholic solvent, such as ethanol or ethanol containing a small amount of 2-mercaptoethanol so as to prevent any oxidation of the resulting compound of formula (I), in the presence of a base, such as sodium hydroxide. In the event that one or both or R² and R³ in the compound of formula (II) is or are C1-4 alkyl, then the conditions preferred for deacylation are also likely to lead to deesterification. In such circumstances, the product of the deacylation is a compound of formula (I), wherein R² and R³ are both hydrogen. In order therefore, to prepare a compound of formula (I), wherein one or both of R² and R³ is or are C1-4 alkyl, the resulting compound of formula (I), wherein R² and R³ are both hydrogen, is esterified as described hereinafter.
- The optional formylation of the resulting compound of formula (I), wherein R¹ is hydrogen, is, preferably, carried out by reacting the compound with acetic formic anhydride at an elevated temperature.
- Examples of the optional conversion of R² and/or R³ in the resulting compound of formula (I) into another R² and/or R³ include the optional conversion of hydrogen into C1-4 alkyl using conventional esterification reagents and conditions, for example a C1-4 alkanol in the presence of an acid. This may be a useful optional conversion to carry out in the event that, in the preparation of a compound of formula (I), wherein one or both of R² and R³ is or are C1-4 alkyl, the compound undergoes deesterification as described hereinbefore. In such circumstances, the desired ester may be prepared from the free acid as just described.
- The optional formation of a salt of a compound of formula (I) may be carried out conventionally.
- The compounds of formula (II) may be prepared by reducing a mixture of a compound of formula (III):
- The reduction of a mixture of the compounds of formulae (III) and (VI) may be carried out in accordance with conventional reductive alkylation reactions. It is however, preferred that the reduction is effected with sodium cyanoborohydride in a solvent in the presence of an acid, such as acetic acid which may also function as the solvent.
- The optional alkylation of the resulting compound of formula (II), wherein R¹ is hydrogen, is, preferably, carried out by reductive alkylation in which a mixture of the compound of formula (II) and a C1-4 aldehyde are reduced with sodium cyanoborohydride in the presence of an acid, such as acetic acid.
- The compounds of formula (II), wherein n is 3, may also, although less preferably, be prepared by reducing a mixture of a compound of formula (IV):
- The reduction of a mixture of the compounds of formulae (IV) and (VI) may be carried out analogously to the reduction of a mixture of the compounds of formulae (III) and (VI) in which sodium cyanoborohydride is used in the presence of an acid, such as acetic acid, to effect the reduction. It is, however, preferred, in the reduction of the mixture of the compounds of formulae (VI) and (IV), that acetic acid is not used also as the solvent but that, for example, methanol or a mixture of methanol and 2-methoxyethanol is used in this regard. It is also preferred that the reduction is carried out in the presence of a dehydrating agent, such as molecular sieves.
- The optional alkylation of the resulting compound of formula (II), wherein R¹¹ is hydrogen, may be carried out as described hereinbefore.
-
- In the case of the preparation of a compound of formula (III), the hydrolysis may be carried out in a solvent, such as dichloromethane or acetone, in the presence of an acid, such as oxalic acid or 4-toluenesulphonic acid. In addition, in the case of the preparation of a compound of formula (III), wherein n is 3, the hydrolysis may also be carried out simply in water at room or a slightly elevated temperature.
- In the case of the preparation of a compound of formula (IV), the hydrolysis is carried out with a compound of formula (V), wherein n is 3, and may be achieved simply in boiling or nearly boiling water.
-
- The acylation of a compound of formula (VII) may be carried out conventionally using, for example, an acid anhydride in the presence of a base, such as pyridine.
-
- The reaction between the compound of formula (VIII) and guanidine may be carried out conventionally, for example, in a solvent, such as ethanol, in the presence of a base, such as sodium methoxide, at an elevated temperature.
-
- The reaction between the compounds of formulae (IX) and (X) may be carried out conventionally, for example, in a solvent, such as ethanol, in the presence of a base, such as sodium methoxide.
- The compounds of formulae (VI), (IX) and (X) are commercially available, or may be obtained by carrying out a published process for their preparation, or by carrying out a process analogous to a published process for the preparation of structurally analogous compounds. For example, the compounds of formula (VI) may be obtained by using the process described in J. Am. Chem. Soc., 1958, 80, page 5778 et seq.
- The compounds of formulae (II), (III), (IV) and (V), on the other hand, are novel intermediates of use in the preparation of the compounds of formula (I) and, thus, represent part of the present invention.
- Compounds of the formula (I) where m is 1 or 2 may be prepared enzymatically from the corresponding compound of the formula (I) where m is 0 by reaction with glutamic acid in the presence of a suitable enzyme, such as E.coli folylpoly-γ-glutamate synthetase.
- While it is possible for the compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, for human or veterinary application, which comprises a compound of the present invention or a pharmaceutically acceptable salt thereof, as hereinbefore defined, and a pharmaceutically acceptable carrier thereof.
- The pharmaceutical formulation may optionally contain other therapeutic agents that may usefully be employed in conjunction with the compound or salt of the present invention, for example a dihydrofolate reductase inhibitor that is capable of enhancing the antineoplastic activity of the compounds and salts of the present invention. The expression "pharmaceutically acceptable" as used herein in relation to the carrier is used in the sense of being compatible with the compound or salt of the invention employed in the formulation and with any other therapeutic agent that may be present, and not being detrimental to the recipient thererof. The carrier itself may constitute one or more excipients conventionally used in the art of pharmacy that enable the compound or salt of the present invention, and any other therapeutic agent that may be present, to be formulated as a pharmaceutical formulation.
- The pharmaceutical formulations of the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous) and rectal administration although the most suitable route will probably depend upon, for example, the condition and identity of the recipient. The formulations may conventionally be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient, i.e. the compound or salt of the present invention, with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or, a finely divided solid carrier or both, and then, if necessary, forming the associated mixture into the desired formulation.
- The pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units, such as a capsule, cachet, tablet, or lozenge, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid such as a syrup, elixir or a draught, or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion. The formulation may also be a bolus, electuary or paste.
- Generally, a tablet is the most convenient pharmaceutical formulation suitable for oral administration. A tablet may be made by compressing or moulding the active ingredient with the pharmaceutically acceptable carrier. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, in admixture with, for example, a binding agent, an inert diluent, a lubricating agent, a disintegrating agent and/or a surface active agent. Moulded tablets may be prepared by moulding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tables may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient.
- The pharmaceutical formulations of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain, for example, a anti-oxidant, a buffer, a bacteriostat and a solute which renders the composition isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may contain, for example, a suspending agent and a thickening agent. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- The pharmaceutical formulations of the present invention suitable for rectal administration may be presented as a suppository containing, for example, cocoa butter and polyethylene glycol.
- As mentioned hereinbefore, the compounds and salts of the present invention have anti-neoplastic activity as demonstrated hereinafter in the lymphocytic leukemia P388/0 and the cell culture cytotoxicity tests, in which a representative number of the compounds of the present invention is shown to be active against lymphocytic and connective tissue cell lines. It has thus been established that the compounds of the present invention are able to inhibit neoplastic growth. Therefore, the compounds and salts of the present invention are of use in human and veterinary medicine and in particular in the treatment of prophylaxis of neoplastic growth, especially lymphocytic leukemia and malignant tumours (otherwise known generally as cancers). Accordingly, the present invention yet further provides a method for the treatment or prophylaxis of neoplastic growth in an animal, which comprises administering to the animal a therapeutically or prophylactically effective amount of a compound or salt of the present invention. In the alternative, there is also provided a compound or salt of the present invention for use in human or veterinary medicine and in particular for use in the treatment or prophylaxis of a neoplastic growth.
- The animal requiring treatment or prophylaxis with a compound or salt of the present invention is usually a human or non-human mammal.
- Particular examples of a neoplastic growth requiring treatment or prophylaxis include lymphocytic leukemia and malignant tumours.
- As mentioned hereinbefore, the antineoplastic activity of the compounds and salts of the present invention may be enhanced by a dihydrofolate reductase inhibitor, for example 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine hydrochloride. Therefore, it may be advantageous to employ with the compounds and salts of the present invention a dihyrofolate reductase inhibitor in the treatment or prophylaxis of neoplastic growth.
- The route by which the compound or salt of the present invention is administered to the animal may be oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous or rectal). If the compound or salt is presented in the form of a pharmaceutical formulation, which, as mentioned hereinbefore, is preferred, then the actual formulation employed will of course depend on the route of administration elected by the physician or veterinarian. For example, if oral administration is preferred, then the pharmaceutical formulation employed is, preferably, one which is suitable for such a route.
- A therapeutically or prophylactically effective amount of a compound or salt of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment or prophylaxis and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. However, an effective amount of a compound of the present invention for the treatment or prophylaxis of neoplastic growth, in particular lymphocytic leukemia or a malignant tumour, will generally be in the range of 0.5 to 600 mg/kg body weight per day. Thus, for a 70kg adult patient, the actual amount per day would usually be from 490 to 14,000mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt of the present invention may be determined as a proportion of the effective amount of the compound per se.
- The treatment or prophylaxis of neoplastic growth with a compound of the present invention may at times require the administration to the animal of an antidote or rescue agent. Particular examples of such agents include leucovorin, hypoxanthine and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), although leucovorin is the most preferred.
- The compounds of Examples 1, 2 and 3 hereinafter have also been found to have activity against the mycoplasma, Spiroplasma citri. These compounds are, therefore, useful in the treatment or prophylaxis of citrus plants that are infected with this microorganism. The compounds may be applied to the plants by methods known in the art, such as spraying, dusting, incorporating into the soil or injecting into the plant. The compound of Example 1 has also been found to possess activity against certain bacterial organisms that are unable to synthesize their own folic acid and that, therefore, require preformed folate. These organisms with the Lactobacillus casei and Streptococcus faecium. And together with the compound of Example 3, the compound of Example 1 has further been found to possess some activity against yellow fever virus.
- The following examples and biological data are provided in illustration of the present invention and should not be construed as in any way constituting a limitation thereof.
- To a stirred solution of 16.2 g (0.30 mol) of sodium methoxide in 100 ml of absolute ethanol was added 160 ml (170 g, 1.50 mol) of ethyl cyanoacetate. The solution was spin evaporated in vacuo at 40°C and the residual white solid was dissolved in 200 ml of dry dimethylformamide. To this solution was added 50 ml (50 g, 0.30 mol) of 3-chloropropionaldehyde diethyl acetal and a crystal of sodium iodide, and the solution was heated on a steam bath with magnetic stirring and protection from moisture for 5 hours. The red-brown solution was cooled, poured into 300 ml of ice water and extracted with diethylether (6 x 200 ml). The organic phase was washed with water (3 x 50 ml), brine (50 ml) and dried over magnesium sulphate. The solution was filtered, spin evaporated in vacuo, and the residue was distilled to give 41.3 g (57%) of a clear liquid, bp 90-108° (0.05 mm Hg) which was sufficiently pure for the next step. Fractional distillation gave as a main fraction a clear liquid, bp 103-108° (0.025 mm Hg); NMR (CDCl₃) δ 1.20 (t, 6H, CH(OCH₂CH₃)₂), 1.31 (t, 3H, CO₂CH₂CH₃), 1.90 (m, 2H, CH₂CHCN), 3.60 (m, 5H, CH(OCH₂CH₃)₂ + CHCN), 4.24 (q, 2H, CO₂CH₂), 4.51 (t, 1H, CH(OCH₂CH₃)₂); 1R (film) 2265, 1750, 1450 cm⁻¹.
- To a solution of 22.0 g (407 mol) of sodium methoxide in 400 ml of absolute ethanol was added 18.2 g (191 mmol) of guanidine hydrochloride and 46.0 g (189 mmol) of ethyl 2-cyano-5,5-diethoxypentanoate. The mixture was refluxed with stirring for 2.5 hours, stirred at ambient temperature overnight and then neutralized with 15 ml of acetic acid. The salts were removed by filtration and washed with 100 ml of ethanol. The combined filtrate and wash were spin evaporated in vacuo to give an off-white solid which was digested with ethyl acetate and cooled. The solid was collected and washed with ethyl acetate. This material, which contained sodium acetate, was dissolved in 150 ml of 1 N sodium hydroxide and then acidified with stirring to pH 5-6 with 10 ml of acetic acid. The resultant precipitate was collected, washed with 50 ml of cold water (product partly soluble in water) and dried; yield, 29.07 g (60%), mp 178-181°. Recrystallization of a portion from ethyl acetate-ethanol gave the analytical sample, mp 177-178°; TLC (C₆H₆: EtOH/5:1); NMR (DMSO-d₆) δ 1.11 (t, 6H, CH₃), 1.56 (m, 2H, CH₂CH₂ CH), 2.19 (t, 2H, CH₂CH₂CH), 3.2-3.7 (m, 4H, 2 x OCH₂), 4.45 (t, 1H, CH), (s, 2H, NH₂, 5.98 (s, 2H, NH₂), 9.84 (s, 1H, HNC(O)).
Elemental analysis: Calculated for C₁₁H₂₀N₄O₃: C, 51.6; H, 7.87; N, 21.9. Found: C, 51.7; H, 7.79; N, 21.7 - A stirred mixture of 7.20 g (28.1 mmol) of 3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-propionaldehyde diethyl acetal, 30 ml of dry pyridine and 30 ml of freshly distilled acetic anhydride was heated on an oil bath at 90° for 6 hours and then stirred at ambient temperature overnight. Solution occurred within 15 minutes as a mixture of diacetyl and triacetyl pyrimidinone formed; extended reaction was required to obtain only the title compound. The solution was spin evaporated in vacuo to give an oil. Ethyl acetate was added and spin evaporated several times until a solid was obtained. The solid was dispersed in cyclohexane and collected; yield, 8.88 g (82%), mp 126-137° (one spot on TLC). Recrystallization of a portion from cyclohexane-ethylacetate gave the analytical sample, mp 138-139°; TLC (C₆H₆: EtOH/5:1); NMR (DMSO-d₆) δ 1.08 (t, 6H, 2CH₂CH₃), 1.63 (m, 2H, CH₂CH₂CH), 2.13 (s, 3H, Ac), 2.27 (s, 6H, 2Ac), 2.2 (2H, CH₂CH₂CH, superimposed on the acetyl singlets), 3.2-3.7 (m, 4H, 2 x OCH₂), 4.42 (t, 1H, CH), 11.87 (br s, 2H, AcNH and HNC(O)).
Elemental analysis: Calculated for C₁₇H₂₆N₄O₆: C, 53.4; H, 6.85; N,14.6. Found: C, 53.7; H, 6.87; N, 14.6. - 3-(2-Acetylamino-4-diacetylamino-1, 6-dihydro-6-oxo-5-pyrimidinyl)-propionaldehyde diethyl acetal (25.0 g, 0.66 mol) was heated with 100 ml of water on a steam bath for 4 hours. The solution was filtered hot to remove an insoluble by-product, and the filtrates were spin evaporated in vacuo to a thick syrup. This syrup was successively dissolved in ethanol and then ethylacetate followed by spin evaporation each time to give a solid which was digested with ethyl acetate. The solids were collected and dried; yield, 15.1 g (76%), mp 199-201° of a cream colored solid which was used without further purification. Recrystallization of a portion from 2-propanol gave tha analytical sample, mp 202-205°; TLC (C₆H₆: EtOH/10.1); NMR (DMSO-d₆) δ 11.75 (br s, 1H, NH), 11.30 (br s, 1H, NH, W1/2 = 5 Hz), 6.15 (d, 1H, J = 5.0 Hz, OH), 6.06 (m, 1H, J = 2.7 Hz when D₂O exchange removed OH CH), 2.43 (s, 3H, CH₃), 2.17 (s, 3H, CH₃), 2.40 - 2.10 (m, 2H, pyrimidine-CH₂), 2.02 - 1.42 (m, 2H, CH₂CH₂CH); ¹³C-FT nmr (DMSO- d₆) ppm: 15.7, 23.9, 27.2, 72.8, 102,2, 147.2 152.4, 160.4, 171.0, 173.4; MS m/e 266, 248.
Elemental analysis: Calculated for C₁₁H₁₄N₄O₄: C, 49.6; H, 5.30; N, 21.04. Found: C, 49.6; H, 5.36; N, 20.35 - A mixture of 2.30 g (7.6 mmol) of 2-acetamido-8-acetyl-5,6,7,8-tetrahydro-7-hydroxypyrido[2,3-d]pyrimidin-4(3H)-one, 2.40 g (8.15 mmol) of dimethyl N-(4-aminobenoyl)-L-glutamate, 5 g of 3A molecular sieves, 150 ml of methanol, and 4 ml of acetic acid was stirred with protection from moisture for 3 hours when "imine" formation was complete. Sodium cyanoborohydride (0.38 g) and 8 ml of acetic acid was added to the reaction. After 15 hours a trace of intermediate "imine" was detected by TLC and an additional 50 mg of sodium cyanoborohbydride was added. After 1 hour the reaction was diluted with 500 ml of chloroform and filtered through a Celite pad to removed the insolubles. The chloroform solution was washed with four 50 ml portions of 5% aqueous sodium bicarbonate, two 50 ml portions of water, one 50 ml portion of brine, dried over magnesium sulphate and spin evaporated in vacuo. The resultant foam was triturated with 50 ml of ethyl acetate to give white crystals; yield, 2.75 g (65%), mp 144- 150°. Recrystallization from ethanol gave chromatographically pure material; yield, 1.70 g (40%), mp 135-142°. An analytically pure sample was obtained from a previous run with mp 140-143°; TLC (C₆H₆: EtOH/10:1); NMR (DMSO-d₆) δ 11.69 (br s, 2H, 2AcNH) 9.68 (br s, 1H, ring NH), 8,24 (br d, 1H, ArCONH), 7.65 (d, 2H, ArH), 6.53 (d, 2H, ArH), 6.17 (br t, 1H, CH₂NH), 4.39 (m, 1H, CHCO₂CH₃), 3.63 (s, 3H, CO₂CH₃), 3.58 (s, 3H, CO₂CH₃), 3.01 (m, 2H, CH₂NHAr), 2.43 (m, 2H, CH₂CO₂CH₃), 2.1 (4H, pyrimidine -CH₂ and CHCH₂, masked by AcNH peaks), 2.14 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.72 (m, 2H, CH₂CH₂CH₂).
Elemental analysis: Calculated for C₂₅H₃₂N₆O₈.1/2 H₂O: C, 54.2; H, 6.01; N, 15.2. Found: C, 54.3; H, 5.90; N, 15.3 - A stirred solution of 1.60 g (2.89 mmol) of dimethyl N-[4-[3-(2,4-bis(acetamido)-1,6-dihydro-6-oxo-5-pyrimidinyl)propylamino]benzoyl]-L-glutamate, 50 ml of ethanol and 100 ml of 1 N sodium hydroxide was heated at 50-60° for 20 hours. The cooled reaction was spin evaporated in vacuo to 50 ml, cooled, and neutralized to pH 5-6 with concentrated hydrochloric acid. The resultant white precipitate was collected, washed with water, diethylether and dried; yield, 0.86 g (66%) mp (sinter 170°) 198-202° (eff). Recrystallization from methanol gave the analytical sample; yield, 0.33 g (39%), mp (forms hard foam 180°) 200° (eff); TLC (pyridine: BuOH:H₂O/1:1:1); NMR (DMSO-d₆) δ 8.07 (d, 1H, ArCONH), 7.65 (d, 2H, ArH), 6.54 (d, 2H, ArH), 6.22 (br s, 1H, CH₂NH), 5.94 (s, 2H, NH₂), 5.75(s, 2H, NH₂), 4.34 (m, 1H, CHCO₂H), 3.03 (m, 2H, CH₂NHAr ), 2.30 (m, 4H, pyrimidine-CH₂ and CH₂CO₂H), 2.03 (m, 2H, CHCH₂), 1.58 (m, 2H, CH₂CH₂CH₂).
Elemental analysis: Calculated for C₁₉H₂₄N₆O₆.H₂O: C, 50.7; H, 5.82; N, 18.7. Found: C, 50.4; H, 5.79; N, 18.6. - Method 1: A solution of 3-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propionaldehyde diethyl acetal (1.00 g, 2.6 mmol) in distilled water (30 ml) was stirred at room temperature for 18 hours. This solution was extracted with ethyl ether (3 x 50 ml), and chloroform (5 x 50 ml). The chloroform extracts were combined, washed with water (25 ml), brine (25 ml), dried (MgSO₄) and spin evaporated in vacuo. The residue was recrystallized from chloroform/cyclohexane to yield 0.200 g (25% of theory) of analytically pure 3-(2-acetylamino-4-diacetyl-amino-1,6-dihydro-6-oxo-5-pyrimidinyl)propionaldehyde quarter hydrate, mp 164-168°C.
- Elemental analysis: Calc for C₁₃H₁₆N₄O₅ 1/4 H₂O (MW 312.803): C, 49.9; H, 5.32; N, 17.9. Found: C, 49.8; H, 5.30; N, 17.6.
- Method 2: A solution of 3-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propionaldehyde diethyl acetal (30.0 g, 78.5 mmol) in distilled water (600 ml) was stirred at 53°C for 3.5 hours and spin evaporated in vacuo at 45°C. The residue was dissolved in dichloromethane (750 ml), washed with brine (50 ml), dried (MgSO₄), and spin evaporated in vacuo to a dry solid.
- Recrystallization from acetone gave 16.23 g (67% of theory) of 3-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl) propionaldehyde, mp 164-168°C, which was identical to that prepared by Method 1.
- To a solution of 3-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propionaldehyde (9.30 g, 30.2 mmol) and dimethyl N-(4-aminobenzoyl)-L-glutamate (9.85 g, 33.5 mmol) (R. Koehler, L. Goodman, J. DeGraw, and B.R. Baker, J. Am. Chem. Soc., 80 5779 (1958) in methanol (175 ml) was added acetic acid (7.7 ml) and 3A molecular sieves (activated at 190°C for 18 hours in vacuum). After stirring for 1 hour under a CaCl₂ drying tube, sodium cyanoborohydride (2.0 g, 31.9 mmol) was added in small portions over 2 minutes. After an additional 3 hours of stirring the reaction was filtered and the sieves were washed with methanol. The methanol was spin evaporated in vacuo to give a residual foam. This foam was added to a column (40mm x 180 mm) of Silica Gel 60 wetted with ethyl acetate and eluted using the "flash chromatography technique" (W.C. Still, M. Kahn, and A Mitra, J. Org. Chem., 43, 2923 1987). The column was washed with 1 litre of ethyl acetate followed by methanol/dichloromethane (1:9) to elute the product. The fractions containing product were combined and spin evaporated in vacuo to yield 8.2 g of the acetylated ester intermediate, mp 175-180°C. A solution of 7.2 g of this intermediate was dissolved in aqueous sodium hydroxide (1N, 500ml) and ethanol (250 ml) and stirred at 55°C for 18 hours. The reaction solution was concentrated to 100 ml by spin evaporation in vacuo. The pH was adjusted to 4.0 with concentrated hydrochloric acid (12N). After cooling, the precipitate was collected and washed with water and ether. The product was sucked dry to give 6.0 g (52%) of crude product that was recrystallised by dissolution in 2-methoxyethanol (75 ml), filtration and slow addition to ethanol (200ml) with stirring to give 1.02 g of N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl]propylamino]benzoyl-L-glutamic acid, mp 195-200°, which was identical to that prepared via the compound of formula (V).
- To a stirred solution of 1.99 g (3.6 mmol) of dimethyl N-[4-[3-(2,4-bis(acetamido)-1,6-dihydro-6-oxo-5-pyrimidinyl) propylamino]benzoyl]-L-glutamate in 50 ml of acetonitrile and 5 ml of dimethylformamide was added 2 ml of 37% aqueous formaldehyde followed by 0.50g (8.0 mmol) of sodium cyanoborohydride and 2 ml of acetic acid. After 18 hours the mixture was filtered to remove the solids and the filtrate was spin evaporated in vacuo. The residue was diluted with 100 ml of ice water, and then extracted with four 25 ml portions of methylene dichloride. The combined extracts were filtered through glass wool and spin evaporated in vacuo to give 2.1 g of the title compound as an oil which was a single spot on TLC. The oil was dissolved in 50 ml of ethanol and 50 ml of 1N sodium hydroxide and stirred at 70°C for 20 hours. The reaction was cooled and spin evaporated in vacuo to a syrup. The syrup was dissolved in 50ml of water, cooled on ice, and acidified to pH 3-4 with concentrated hydrochloric acid. The white solids were collected, washed with water, and finally with ether; yield, 1.10 g (67%), mp 190- 200°C (one spot on TLC). Recrystallization from ethanol gave the analytical sample; yield, 0.54 g (33%), mp (166-168°C changes to hard foam) 198-204°; TLC (pyridine: BuOH:H₂O/1:1:1); NMR (DMSO-d₆) δ 8.16 (d, 1H, ArCONH), 7.71 (d, 2H, Ar), 6.66 (d, 2H, Ar), 5.94 (s, 2H, NH₂), 5.76 (s, 2H, NH₂) 4.36 (m, 1H, CHCO₂H), 3.34 (m, 2H, CH₂N (CH ₃)Ar, 2.93 (s, 3H, NCH₃).
Elemental analysis: Calculated for C₂₀H₂₆N₆O₆. 1/2 H₂O: C, 52.7; H, 5.98; N, 18.4. Found: C, 52.6; H, 5.95; N, 18.4. - A solution of 1.16 g (2.5 mmol) of N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propylamino]benzoyl]-L-glutamic acid in the solution prepared from 25 ml of 97% formic acid and 5 ml of acetic anhydride was heated on an oil bath at reflux for 1 hour. The reaction was cooled and spin evaporated in vacuo to give a hard foam, which was repeatedly covered with ethanol and reevaporated. The foam was triturated under ethanol with a magnetic stirring bar overnight to give a fine solid which was collected, washed with ethanol, and sucked dry; yield, 1.22 g (98%), mp 130-160° (eff). Several recrystallizations from ethanol gave analytically pure material; yield, 0.496 g (40%), mp 170-180°C (eff); NMR (DMSO-d₆) δ 8.63 (d, 1H, ArCONH), 8.53 and 8.37 (two s in ratio 9 to 1 at 29°C, coalesce to a singlet at 120°C, 1H, HCON), 7.93 (d, 2H, ArH), 7.56 (s, ArH of one formanilide isomer), 7.44 (d, 2H, ArH), 5.93 and 5.70 (two s, 4H, two NH₂), 4.41 (m, 1H, CHCO₂H), 3.78 (m, 2H, CH₂N (CHO)Ar).
Elemental analysis: Calculated for C₂₀H₂₄N₆O₇, H₂O: C, 50.2; H, 5.48; N, 17.6. Found: C, 50.2; H, 5.16; N, 17.9. - Freshly distilled acetic anhydride (250 ml) was added to a solution of 2-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl) acetaldehyde diethyl acetal (Chem.. Ber., 1977, 110, 1462) (50.0 g 0.206 mol) in dry pyridine (250 ml). The solution was protected from moisture and heated on a steam bath with magnetic stirring for 5 days. The reaction was spin evaporated in vacuo with the addition of ethylene glycol monomethyl ether under aspirator and finally mechanical pump vacuum. The residue was recrystallized from ether to yield 23.0 g (30% of theory) of 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-acetaldehyde diethyl acetal, mp 128-130°. The analytical sample was prepared by dissolving the sample in chloroform and washing the solution through Superfiltrol µ19 (Filtrol Corporation). The solution was spin evaporated in vacuo and the residue was recrystallized from ether/cyclohexanes, to give analytically pure material, mp 129-130°C.
Elemental analysis: Calculated for C₁₆H₂₄N₄O₆ (MW 368.40): C, 52.17; H, 6.97; N, 15.20. Found: C, 51.82; H, 6.50; N, 15.25. - Method 1: slurry of Silica Gel 60 (6.0 g, Merck µ7734, 70-230 mesh) and aqueous oxalic acid (10%) (0.6 ml) in dichloromethane (12 ml) was stirred for 15 minutes. 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-acetaldehyde diethyl acetal (1.0 g, 2.7 mmol) was added and the mixture was stirred for 18 hours. Sodium bicarbonate (0.2 g, 2.4 mmol) was added and after 10 minutes the slurry was filtered and washed with ethyl acetate. The filtrates and wash were combined and spin evaporated in vacuo to give 0.37 g (46% of theory) of 2-(2-acetylamino-4-diacetylemino-1,6-dihydro-6-oxo-5-pyrimidinyl)acetaldehyde. The analytical sample was prepared by recrystallization from acetone to give 0.21 g (26% of theory), mp 169-170°C)
Elemental analysis: calculated for C₁₂H₁₄N₄O₅ (MW 294.27): C, 48.98; H, 4.80; N, 17.08. Found C, 48.90; H, 4.83; N, 19,26. - Method 2: A solution 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)acetaldehyde diethyl acetal (2.0 g, 5.4 mmol) and 4- toluenesulfonic acid monhydrate (0.050 g, 0.26 mmol) in acetone (100 ml) was stirred for 2 days. The solids were collected, washed with acetone and dried to give 0.73 g (46% of theory) of 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)acetaldehyde, mp 168-170° which was identical to that prepared by method 1.
- A solution of 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)acetaldehyde (1.74 g, 5.92 mmol) and dimethyl N-(4- aminobenzoyl)-L-glutamate (J. Am. Chem. Soc., 1958, 80, 5779) (1.74 g, 5.92 mmol) in acetic acid (90 ml) was stirred for 10 minutes. Sodium cyanoborohydride (0.84 g, 13.4 mmol) was added in small portions over 1 hour. After an additional 30 minutes, the reaction was spin evaporated in vacuo to a thick oil. The oil was dissolved in ethyl acetate (500 ml) and washed with 5% aqueous sodium bicarbonate until neutral, water (50 ml), brine (50 ml), dried with magnesium sulfate and spin evaporated in vacuo. Recrystallization from ethyl acetate, with filtration to remove insolubles, yielded 1.5 g (49% of theory) of dimethyl N-[4-[2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethylamino]benzoyl]-L-glutamate, mp 168-178°C. Recrystallization from ethyl acetate (Norite) gave 0.830 g (25% of theory) of the analytical sample, mp 176-179°C.
Elemental analysis: Calculated for C₂₆H₃₂N₆O₉.H₂O (MW 590.60: C, 49.79; H, 5.68: N, 18.93. Found: C, 49.62; H, 5.53; N, 18.87. - A crude preparation of dimethyl N-[4-[2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethylamino]benzoyl]-L-glutamate hydrate (prepared on a 3.3 mmol scale but without recrystallization) was dissolved in 1N sodium hydroxide (100 ml) and ethanol (50 ml) containing 2-mercaptoethanol (0.25 ml). The reaction was heated with stirring to 55° for 18 hours and then spin evaporated in vacuo to a small volume. The pH of the ice bath cooled solution was adjusted to pH 4 with hydrochloric acid (12 N) to precipitate the product. Recrystallization from methanol/ether gave 0.52 g (38% of theory) of N-[4-[2-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethyl amino]benzoyl]-L-glutamic acid, mp 180-185°.
Elemental analysis: Calculated for C₁₈H₂₂N₆O₆,H₂O.0.1 Et₂O (MW 443.84): C 49.79; H, 5.68; N, 18.92. Found: C, 49.62; H, 5.53; N, 18.87. - A solution of 2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-5-pyrimidinyl)acetaldehyde (2.93 g, 10 mmol) and dimethyl N-(4-aminobenzoyl)-L-glutamate (2.93 g, 10 mmol) in acetic acid (200 ml) was stirred at ambient temperature for 15 minutes. Sodium cyanoborohydride (0.350 g, 5.6 mmol) was added, and after an additional 10 minutes aqueous formaldehyde (37%) (4.2 ml) was added. After 10 minutes additional sodium cyanoborohydride (1.5 g, 23.0 mmol) was added and the reaction was stirred for 30 minutes. The solvent was removed by spin evaporation in vacuo at 50°C. The viscous oil was dissolved in chloroform:methanol (1:10, 500 ml) and extracted with water (50 ml), 5% aqueous sodium bicarbonate (10 ml) and dried over magnesium sulfate. After spin evaporation in vacuo the residue was recrystallized from ether to give 3.14 g of a pale yellow powder. The product was purified by open column chromatography on a Silica Gel 60 (Merck µ7734, 63-200 µm) column (3 cm x 25 cm column) wetted with dichloromethane and eluted with 4% methanol in dichloromethane. The appropriate fractions were combined and spin evaporated in vacuo, and the residue was recrystallized from ethyl acetate to yield 1.3 g (22% of theory) of dimethyl N-[4-[2-(2,4-bis(acetamido)-1,6-dihydro-6-oxo-5-pyrimidinyl)-ethyl-N-methylamino]benzoyl-L-glutamate, mp 168-175°.
Elemental analysis: Calculated for C₂₅H₃₃N₆O₈.0.5 H₂O (MW 553.58): C, 54.24: H, 6.01; N, 15.18. Found: C, 53.88; H, 5.75, N, 15.28. - A solution of dimethyl N-[4-[2-(2-acetylamino-4-diacetylamino-1,6-dihydro-6-oxo-pyrimidinyl)ethyl-N-methylamino]benzoyl]-L-glutamate (0.25 g, 0.43 mmol), 2-mercaptoethanol (0.25 ml), 1N sodium hydroxide (20 ml) and ethanol (10 ml) was stirred under nitrogen for 18 hours at 60°C. The reaction was cooled in an ice bath, and the pH of the solution was adjusted to 4 with hydrochloric acid (12 N). The resultant precipitate was collected and recrystallized from ethanol containing 2-mercaptoethanol (0.25 ml) to yield 0.077 g (41% of theory) of N-[4-[2-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethyl-N-methylamino]benzoyl]-L-glutamic acid, m.p. 180-188°. This material was a white powder which became tan upon extended exposure to light.
- Elemental anylysis: Calculated for C₁₉H₂₄N₆O₆.H₂O (MW 450.46): C, 50.66; H, 5.82; N, 18.66. Found: C, 50.49; H, 5.77; 18.52.
-
- Compounds 2 and 3 were enzymatically synthesized from N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propylamino]benzoyl]-L-glutamic acid (1) hydrate, and L-glutamic acid by E.coli folylpoly-γ-glutamate synthetase (Bognar),A.S., Osborne, C., Shane, B., Singer, S.C., and Ferone, R. J. Biol. Chem. 260, 5625- 5630 (1985)). The 20 hr reaction was done in an amber, gas-tight bottle in a gently shaking 37°C H₂O bath. The argon-saturated 20 ml reaction contained 4 #moles ¹⁴C-1 labeled in the benzoyl carbonyl, 40 µmoles L-glutamic acid, 1 mmole KCl, 4mg bovine serum albumin, 1.4 mmoles 2-mercaptoethanol (2-MET), 0.1 mmole adenosine-5ʹ-triphosphate, 0.2 mmole MgCl₂, 2 mmole Tris-HCl pH 8.6 (37°) and 50 units of enzyme. HPLC analysis of 100 µl of the reaction mixture after 20 hr revealed two ¹⁴C-containing products and no remaining ¹⁴C (1).
- The reaction mixture was adjusted to pH 1 with 2.8 ml of 10% wt-vol trifluoroacetic acid (TFA). It was protected from light during all handling. A 50 ml portion was desalted on a Waters C₁₈ Sep-Pak which had been prewashed with 5 ml of 2-propanol (ISP), 10 ml H₂O and 5 ml 0.1% wt/vol TFA. The ¹⁴C- containing products were retained on the Sep-Pak during a 10 ml 0.1% wt/vol TFA wash and eluted with 8 ml 0.1% TFA, 50% ISP 50 #1 2-MET. The eluant was argon saturated in a sealed amber bottle, shell-frozen and lyophilized. The dried sample was dissolved in 1.0 ml 3% acetonitrile (ACN) in HPLC solvent (0.14 M sodium acetate pH 6.4, 0.05% triethylamine) and passed through a 0.45 µm Millipore HV filter. The filtrate (840 µl) was chromatographed on a 10 cm Waters C₁₈ Nova-Pak Radial Compression Column at 1 ml/min (1.5% ACN in HPLC solvent for 15 minutes followed by 2.4% ACN in HPLC solvent). Two ¹⁴C-containing peaks, identified as 3 and 2 were eluted at 27 and 33 minutes, respectively. Samples were adjusted to pH 1, desalted on C₁₈ Sep-Paks, eluted into 2-Met, argon-saturated and freeze-dried as above. Dried samples were each dissolved in 0.5 ml of argon-saturated 500 µM NaOH. Final yield was 191 nmoles 3 and 337 nmoles 2. The sum equals 71% overall yield from 1.
- The identities of 3 and 2 were established by hydrophobic chromatography of the intact molecules and of their KMnO₄-H₂O₂ induced cleavage products. It is known that the oxidation state of pteroylpolyglutamates and close analogues is the primary factor in their retention on C₁₈ at pH 2. Such compounds, with 1 to 7 glutamates, are retained in 0.1% TFA, 4% ACN and elute in a single sharp peak after 684 second in 1 ml/min 0.1% TFA, 12% ACN. Compounds 3 and 2 co-chromatographed with 1 under the above conditions. This showed that the pyrimidinylpropyl moiety of the compounds was the same as in the parent 1.
- The glutamate chain length of 3 and 2 was established after cleavage of the compounds at the propylamino C-N bond. (A 1.0 ml solution at pH 9 was oxidized with 100 µl 2% KMnO₄. After one minute, 80 µl 30% H₂O₂ was added to precipitate KMnO₂. Excess H₂O₂ was destroyed by the addition of 20 µl of 11 µg/ml beef liver catalase, Worthington Biochemical Corp). The ¹⁴C-labeled oxidation products of 3 and 2 co-chromatographed (on C₁₈ in 0.1% TFA, 4% ACN), respectively, with 4-aminobenzoyl-L-glutamyl-L-glutamyl-L-glutamic acid (PABG₃) and 4-aminobenzyl-L-glutamyl-L-glutamic acid (PABG₂). This shows that the compound assigned as 3 contains three glutamic acid residues attached to 4-aminobenzoic acid. The compound assigned as 2 contains two glutamic acid residues attached to 4-aminobenzoic acid. As in the PAGB₂ and PABG₃ standards, the peptide linkages are through the α-carboxyls of the glutamates.
- To a stirred solution of 3.89 g (7.5 mmol) of N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester dicyclohexylamine salt, 2.22 g (7.5 mmol) of L- glutamic acid bis(tert-butyl)ester hydrochloride, and 1.01 g (7.5 mmol) of 1- hydroxybenzotriazole in 20 ml of dichloromethane was added 1.70 g(8.25 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred for 17 hours and filtered. The filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 40 ml), 5% aqueous citric acid (2 x 40 ml) and saturated brine (2 x 40 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo. The product was purified by flash chromotography on silica gel 60 (150 g, 230- 400 mesh, EM Reagents) with hexanes: ethyl acetate (1:1). The appropriate fractions were combined on the basis of TLC correlation and spin evaporated in vacuo to yield 3.34 g (77% of theory) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-glutamic acid tris(tert-butyl)ester as a colorless gum; TLC (Hexanes: ethyl acetate-3:1), Rf=0.4(anisaldehyde); NMR (DMSO-d₆) δ 1.39(s,27H,O-t-Bu), 1.70(m,2H,α-CH₂), 1.85(m,2H,αCH₂), 2.20(m,4H,β-CH₂, 4.00(m,2H,αH), 5.04( s,2H,NCO₂CH₂), 7.35(s,5H,Ar), 7.55(d,1H,NH), 8.05(d,1H,NH).
Elemental analysis: Calculated for C₃₀H₄₆N₂O₉ (MW 578.70): C, 62.27: H, 8.01; N, 4.84. Found: C, 61.99;H, 8.03; N, 4.79. - A solution of 3.34 g (5.77 mmol) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L- glutamic acid tris(tert)-butyl)ester in 100 ml of 95% ethanol and 80 mg of 10% palladium on carbon was shaken in the presence of hydrogen at 22 psi of hydrogen for 18 hours. The catalyst was removed by filtration and the filtrate was spin evaporated in vacuo to yield 2.39 g (95% of theory) of L-gamma- glutamyl-L-glutamic acid tris(tert-butyl) ester as a colorless syrup; TLC (ethyl acetate), Rf=0.3(ninhydrin). The product was used in the next reaction without further characterization.
- To a stirred mixture of 2.79 g (5.38 mmol) of N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester dicyclohexylamine salt, 2.39 g (5.38 mmol) of L- gamma-glutamyl-L-glutamic acid tris(tert-butyl)ester, 0.73 g(5.38 mmol) of 1- hydroxybenzotriazole and 5.38 ml of 1.0 N hydrochloric acid in 30 ml of dichloromethane was added 1.22 g (5.91 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred for 16 hours and filtered. The filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2x50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 50 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo. The product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with ethyl acetate : hexanes (1:1). The appropriate fractions were combined on the basis of TLC correlation and spin evaporated in vacuo to yield 3.10 g (75% of theory) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid tetrakis(tert-butyl) ester, mp 76-78° : TLC (Hexanes:ethyl acetate-1:1), Rf=0.4 (anisaldehyde); NMR (DMSO-d₆) δ1.39(s,36H, O-t-Bu), 1.75 (m, 3H, α- CH₂), 1.91 (m,3H, α-CH)₂, 2.22(m,6H, β-CH₂), 3.90 (m, 1H, α-H), 4.10(m,2H, α- H), 5.04(dd, 2H, NCO₂CH₂), 7.36(s,5H, Ar), 7.61 (d, 1H, NH), 8.09 (m,2H, NH).
- Elemental analysis. Calculated for C₃₉H₆₁N₃O₁₂ (MW 763.92) : C, 61.32; H, 8.05; N, 5.50. Found: C, 61.09; H, 8.21; N, 5.45.
- A solution of 3.10 g (4.06 mmol) of N-benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid tetrakis(tert-butyl)ester in 100 ml of 95% ethanol and 100 mg of 10% palladium on carbon was shaken in the presence of hydrogen at 22 psi for 8 hours. The catalyst was removed by filtration and the filtrate was spin evaporated in vacuo to yield 2.23 g (87% of theory) of L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid tetrakis(tert-butyl) ester as a colorless gum; TLC (95% ethanol), Rf=0.7(ninhydrin). The product was used in the next reaction without other characterization.
- To a stirred mixture of 1.82 g (3.25 mmol) of N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester dicyclohexylamine salt, 2.23 g (3.52 mmol) of L-gamma-glutamyl-L-glutamic acid tetrakis(tert-butyl)ester, 0.474g (3.52 mmol) of 1-hydroxybenzotriazole and 3.52 ml of 1.0 N hydrochloric acid in 25 ml of dichloromethane was added 0.800 g(3.88 mmol) of 1,3- dicyclohexylcarbodiimide. The reaction was stirred for 17 hours and filtered. The filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 50 ml), dried with magnesium sulfate, filtered and spin evaporated in vacuo. The product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with hexanes: ethyl acetate (3:7). The appropriate fractions were combined on the basis of TLC correlation and spin evaporated in vacuo to yield 2.71 g (81% of theory) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid pentakis(tert-butyl)ester, mp 75°; TLC (hexanes; ethyl acetate-3:7), Rf=0.5 (anisaldehyde); NMR (DMSO- d₆) δ1.39 (s, 45H, O-t-Bu), 1.75 (m,4H,α-CH₂), 1.92 (m,4H,α-CH₂), 1.92 (m, 4H,α-CH₂), 1.92 (m, 4H, α-CH₂), 2.22(m, 8H, β-CH₂), 3.89 (m, 1H, α-H), 4.10 (m,3H,α-H), 5.04(d d,2H,NCO₂CH₂),7.37(s,5H,Ar), 7.63 (d,1H,NH),8.0(m,3H,NH).
- Elemental analysis. Calculated for C₁₄₈H₇₆N₄O₁₅(MW 949.15): c, 60.74; H, 8.07; N,5.90. Found: C, 60.91; H, 8.09; N,6.11.
- A solution of 2.71 g (2.86 mmol) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid pentakis(tert-butyl) ester in 100 ml of 95% ethanol and 100 mg of 10% palladium on carbon was shaken in the presence of hydrogen at 40 psi for 19 hours. The catalyst was removed by filtration and the filtrate was spin evaporated in vacuo to yield 2.29 g (98% of theory) of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid pentakis(tert-butyl) ester as a white foam; TLC (ethyl acetate:methanol- 9:1), Rf=0.5(ninhydrin). The product was used in the next reaction without further characterization.
- To a stirred mixture of 1.46 g (2.80 mmol) of N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester dicyclohexylamine salt, 2.29 g (2.80 mmol) of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid pentakis (tert-butyl) ester, 0.380 g (2.80 mmol) of 1-hydroxybenzotriazole and 2.8 ml of 1.0 N hydrochloric acid in 35 ml of dichloromethane was added 0.635 g (3.08 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred for 19 hours and filtered. The filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 25 ml), 5% aqueous citric acid (2 x 25 ml) and saturated brine (2 x 25 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo. The product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents)with hexanes:ethyl acetate (1:2). The appropriate fractions were combined on the basis of TLC correlation and spin evaporated in vacuo to yield 2.40 g (75% of theory) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid hexakis (tert-butyl) ester as a white foam, mp 74-76°; TLC (hexanes:ethyl acetate-1:2), Rf=0.4 (anisaldehyde). Elemental analysis. Calculated for C₅₇H₉₁H₅O₁₈ (MW 1134.37): C, 60.35; H, 8.09; N, 6.17. Found: C, 59.94; H, 8.16; N, 5.89.
- A solution of 2.40 g (2.11 mmol) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid hexakis (tert-butyl) ester in 100 ml of 95% ethanol and 200 mg of 10% palladium on carbon was shaken in the presence of hydrogen at 45 psi for 4.5 hours. The catalyst was removed by filtration. The filtrate was spin evaporated in vacuo to yield 2.14 g (100% of theory of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid hexakis(tert-butyl)ester as colorless gum; TLC (ethyl acetate:methanol-9:1), Rf=0.3 (ninhydrin). The product was used in the next reaction without further characterization.
- To a stirred mixture of 1.11 g (2.14 mmol) of N-(benzyloxycarbonyl)-L- glutamic acid alpha-tert-butyl ester dicyclohexylamine salt, 2.14 g (2.14 mmol) of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid hexakis(tert-butyl)ester, 0.289g(2.14 mmol) of 1-hydroxybenzotriazole and 2.14 ml of 1.0 N hydrochloric acid in 20 ml of dichloromethane was added 0.486 g (2.35 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred for 18 hours and filtered. The filtrate was sequentially washed with saturated aqueous sodium bicarbonate (2 x 50 ml), 5% aqueous citric acid (2 x 50 ml) and saturated brine (2 x 25 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo. The product was purified by flash chromatography on silica gel 60 (150 g, 230-400 mesh, EM Reagents) with hexanes:ethyl acetate (1:3). The appropriate fractions were combined on the basis of TLC correlation and spin evaporated in vacuo to yield 2.03 g (72% of theory) of N(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis(tert-butyl)ester as a white foam, mp 77-80°; TLC (hexanes:ethyl acetate-1:3), Rf=0.4 (anisaldehyde). Elemental analysis. Calculated for C₆₆H₁₀₆N₆O₂₁ (MW 1319-59):C, 60.07; H, 8.10; N, 6.37. Found: C, 60.00; H, 8.11; N, 6.35.
- A solution of 2.00 g (1.52 mmol) of N-(benzyloxycarbonyl)-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis (tert-butyl) ester in 100 ml of 95% ethanol and 100 mg of 10% palladium on carbon was shaken in the presence of hydrogen at 43 psi for 16 hours. The catalyst was removed by filtration and the filtrate was spin evaporated in vacuo to yield 1.67 (93% of theory) of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis(tert-butyl) ester as a white foam, mp 67-69°; TLC (ethyl acetate:methanol-9:1), Rf=0.5 (ninhydrin); NMR (DMSO-d₆)δ 1.39 (s,63H, O-t-Bu). 1.74 (m,6H, α-CH₂), 1.92 (m, 6H, α-CH₂), 2.21 (m,12H, β-CH₂), 4.08 (m, 6H, α-H), 8.15 (m,7H, NH); mass spectrum (chemical ionization) m/z 1186 (85.0% relative abundance; (M⁺ H⁺), m/z 1001 (27.5%), m/z 815 (100%). Elemental analysis. Calculated for C₅₈H₁₀₀N₆O₁₉ (MW 1185.46): C,58.77; H, 8.50; N, 7.09. Found: C, 58.78; H, 8.58; N, 7.03.
- A mixture of 1.25 g (4.00 mmol) of 3-[2-(acetylamino)-1, 6-dihydro-6-oxo-5-pyrimidinyl]propionaldehyde, 0.743 g (4.50 mmol) of ethyl p-aminobenzoate, 1.0 ml of glacial acetic acid, and 3A molecular sieves in 25 ml of methanol was stirred at room temperature for 3 hours under nitrogen a 2 minute period. After stirring for 17 hours, the mixture was filtered. The filtrate was spin evaporated in vacuo to a yellow foam. The product was separated from a mixture by flash chromatography on silica gel 60 (100 g; 230-400 mesh, EM Reagents) with ethyl acetate. Appropriate fractions were combined on the basis of TLC correlation, and solvent was removed by spin evaporation in vacuo. Recrystallization from ethyl acetate yielded 0.44 g (24% of theory) of ethyl[4-[3-[2-(acetylamino)-4-(diacetylamino)-1,6-dihydro-6-oxo-5-pyrimidinylpropyl]amino]benzoate as a white solid, mp 197-198°; TLC (ethyl acetate), Rf=0.5; NMR (DMSO-d₆) δ 1.28 (t,J=7.1 Hz, 3H,CH₃), 1.65 (m,2H,CCH₂C), 2.14 (s, 3Hm Ac), 2.2 (m, 2H, CH₂Het), 2.23 (s,6H, 2Ac), 3.05 (m,2H,CH₂N), 4.22 (q,J=7.1 Hz, 2H, CO₂CH₂, 6.5 (br,1H,AcNH obscured by Ar), 6.57 (d, 2H, ArH), 7.69 (d, 2H, ArH). Elemental analysis. Calculated for C₂₂H₂₇M₅O₆. 0.5 H₂O (MW 466.49):C, 56.64; H, 6.05; N, 15.01. Found: C, 56.88; H, 6.01; N, 14.88.
- To a solution of 0.22g(0.47 mmol) of ethyl 4-[3-[2-(acetylamino)-4-(diacetylamino)-1,6-dihydro-6-oxo-5-pyrimidinyl]propyl]amino]benzoate in 7.0 ml of 95% ethyl alcohol was added 15 ml of 1.0 N NaOH. The reaction was heated at 70°C for 20 hours. The mixture was reduced by spin evaporation in vacuo to a 10-ml volume and adjusted to pH 5 with 1.0 N HCl. The precipitate was collected by filtration, washed with water, and dried in vacuo to yield 0.11 g (71% of theory) of 4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]benzoic acid one-half sodium salt as a white solid, mp 273° (dec.); HPLC on Versapack C-18 (10 micron; 4.6 x 250 mm) with aqueous 60% methanol containing 0.2% trifluoroacetic acid gave one major peak, k¹=0.58; NMR (DMSO-d₆)δ 1.58 (m, 2H, CCH₂C), 2.24 (t, 2H, Het-CH₂), 3.02 (m, 2H, CH₂N), 5.77 (br S,2H, NH₂), 5.94 (br s, 2H, NH₂), 6.46 (t, 1H, NH-Ar), 6.54 (d, 2H, Ar), 7.65 (d,2H,Ar), 9.80 (br s, 1H, NH), 11.95 (br s, 0.5H,CO₂H). Elemental analysis. Calculated for C₁₄H₁₆.₅N₅O₃Na0.5 (MW 327.82);C,51.29 H,16.5 5.53, N 21.36; Na, 3.51. Found: C, 51.34; H, 5.39; N, 21.32; Na, 3.33.
- A mixture of 0.50 g (153 mmol) of 4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]benzoic acid one-half sodium salt 0.75 water and 5,0 ml trifluoroacetic anhydride was stirred at ambient temperature for 18 hours under nitrogen. The amber solution was spin evaporated in vacuo at 25°. The residual foam was triturated with water (15 ml) until a homogenous, beige powder was obtained. The solid was collected by filtration, "washed with water (2 x 2 ml) and dried in vacuo to yield 0.617 g (81% of theory) of 4-[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]trifluoroacetamido]benzoic acid, mp 229-230°; HPLC on Versapack C-18 (10 micron; 4.6 x 250 mm) with aqueous 50% methanol containing 0.1% trifluoroacetic acid gave one major peak, K¹= 1.00; TLC (methanol:ethyl acetate-1:1), Rf=0.5; NMR (DMSO- d₆)δ1.50 (m, 2H, CCH₂C), 2.20 (t, 2H, Het-CH₂), 3.74 (t,2H,CH₂N), 6.67 (br s, 2H, NH₂), 7.43 (br, 2H, NH₂), 7.55 (d, 2H, Ar), 8.02 (d, 2H, Ar); mass spectrum (methane chemical ionization) m/z 400 (18.5% relative abundance: [M + H]+), 356 (5.91%, [M -CO₂]⁺), 167 (10.72%), 115 (100%). Elemental analysis. Calculated for C₁₆H₁₆F₃N₅O₄. CF₃COOH (MW 513.353): C, 42.11; H, 3.34; N, 13.64. Found: C, 42.47; H, 3.49; N, 14.13.
- To 0.10g (0.20mmol) of 4-[[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]trifluoroacetamido]benzoic acid, 0.24 g (0.20 mmol) of L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-<E L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis(tert-butyl) ester, 0.027 g (0.20 mmol) of 1- hydroxybenzotriazole, and 0.020 g (0.20 mmol) of triethylamine in 2.0 ml of N,N-dimethylformamide at ambient temperature was added 0.046 g (0.22 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred for 22 hours and filtered. The filtrate was spin evaporated in vacuo, and the amber residue was partitioned between dichloromethane (25 ml) and saturated aqueous sodium bicarbonate (15 ml). The organic layer was sequentially washed with saturated sodium bicarbonate (15 ml) and saturated brine (2 x 10 ml), dried with magnesium sulfate, filtered, and spin evaporated in vacuo to a foam.
- The residue was mixed with 5 ml of ethyl acetate; a small amount of insoluble solid was removed by filtration. The filtrate was spin evaporated in vacuo to yield 0.30 g (96% of theory) of N-[4-[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]trifluoroacetamido]benzoyl] -L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis(tert-butyl)ester as a beige solid, mp 114° (dec.); HPLC on Supelco LC-8 with aqueous 80% methanol containing 0.1% triethylamine gave one major peak, k¹=3.35; TLC (methanol:ethyl acetate-1:9), Rf=0.3 (UV and anisaldehyde); NMR (DMSO-d₆) δ 1.38 (m,63H, 0-t-Bu), 1.60 (m, 2H, CCH₂C), 1.72 (m, 6H, α-CH₂), 1.90 (m, 6H,α-CH₂), 2.22 (m, 14H, Het-CH₂ and β-CH₂), 3.71 (t, 2H, CH₂N), 4.07 (m, 5H, α-H), 4.30 (m, 1H, ArCO₂NHCH), 5.72 (s, 2H, NH₂), 5.90 (s. 2H, NH₂), 7.57 (d, 2H, Ar), 7.94 (d, 2H, Ar), 8.13 (m, 6H, α-NH), 9.7 (br, 1H,NH). Elemental analysis. Calculated for C₇₄H₁₁₄F₃N₁₁O₂₂ (MW 1566.77): C, 56.73; H, 7.33; N, 9.83. Found: C, 56.86; H, 7.42; N, 9.79.
- To a solution of 0.30 g (0.20 mmol) of N-[4-[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]trifluoroacetamido]benzoyl]-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis (tert--butyl)ester in 4.0 ml of methanol at ambient temperature was added sequentially 0.045g (1.0 mmol) of dimethylamine and 0.4 ml of water. Under nitrogen,the yellow solution was stirred at ambient temperature for 18 hours. Reaction progress was monitered by HPLC. The solvent was removed by spin evaporation in vacuo to yield 0.30 g (100% of theory) of N-[4-[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]benzoyl- as a beige solid; HPLC on Supelco LC-8 with aqueous 80% methanol containing 0.1% triethylamine gave one major peak, K¹=2.44; TLC (methanol:ethyl acetate-1:4), Rf=0.6 (UV and anisaldehyde); NMR (DMSO-d₆)δ 1.38 (m, 63H, 0-t-Bu), 1.60 (m, 2H, CCH₂C), 1.70 (m,6H, α-CH₂), 1.90 (m, 6H, α-CH2), 2.20 (m, 14H, Het-CH₂ and β-CH₂), 3.00 (m, 2H, CH₂),4.08 (m, 5H, α-H), 4.26 (m, 1H, ArCO₂NHCH), 5.75 (s, 2H, NH₂), 5.91 (s, 2H, NH₂), 6.23 (br, 1H, ArNH), 6.52 (d, 2H, Ar), 7.65 (d, 2H,Ar), 8.14 (m, 6H, β-NH), 9.7 (br, 1H, NH).
- A mixture of 0.30 g (0.20 mmol) of N-[4-[N-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]benzoyl]-L-gamma-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamyl-L-gamma-glutamyl-L-glutamic acid heptakis (tert-butyl)ester in 5.0 ml of trifluoroacetic acid was stirred at ambient temperature for 0.75 hour. The resulting yellow solution was spin evaporated in vacuo to a beige solid, which was subsequently dissolved in 5.0 ml of water. The aqueous solution was injected into a column of C-18 (comprised of a series of five Rainin Spice cartridges connected in series preequillibrated with water. The column was washed with 50 ml of water before the product was eluted with 20% acetonitrile. Solvent was removed by spin evaporation and freeze-drying. The residue was partitioned between a 1:1 mixture of ethylacetate and water. The aqueous layer was freeze-dried to yield 0.12 g (52% of theory) of N-[4-[N-[3-(2,4-diamimo-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]benzoyl]-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-gamma-glutamyl-L-glutamic acid acid as a fluffy white powder, mp 204°(dec.). HPLC on Versapack C-18 (10 micron; 4.6 x 250 mm) with aqueous 15% acetonitrile containing 0.2%CF₃CO₂H gave one major peak, k¹=0.77. 1H NMR (DMSO) δ 1.60 (m, 2H, CCH₂C), 1.73 (m, 6H, α-CH₂), 1.95 (m, 6H, α-CH₂), 2.19 (m, 14H, Het-CH₂ and β-CH₂), 3.00 (m, 2H, CH₂N), 4.13 (m, 5H, α-H), 4.30 (m, 1H, ArCO₂NHCH), 5.77 (s, 2H, NH₂), 5.95 (s, 2H, NH₂), 6.20 (br, 1H, ArNH), 6.53 (d, 2H, Ar), 7.68 (d, 2H, Ar), 8.15 (m, 6H, α-NH), 12.4 (br, COOH signals) plus 0.25 mol EtOAc(1.18 t, 2.00 s, 4.03 q). Elemental analysis: Calculated for C₄₄H₅₉N₁₁O₂₁. 2.5 H₂O.0.25 EtOAc (MW 1145.08): C, 47.20; H, 5.81; N, 13.46. Found: C, 47.35; H, 5.61; N, 13.20.
- The tests employed for the evaluation of anti-neoplastic activity of the compounds of the present invention are essentially those used in the Tumor Panel of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, A. Goldin, et al., Methods in Cancer Research, Vol. XVI, p. 165, Academic Press (1979). Some modifications, in dose level and schedule have been made to increase the testing efficiency.
- CD2-F₁-mice, of the same sex, weighing withi a 3 g range surrounding 20 g, are used for this test. Control and test animals are injected intraperitoneally with a suspension of 106 viable P388/0 tumor cells on day 0. In each test, several dose levels which bracket the LD₂₀ of the compound are evaluated; each dose level group contains six animals. The test compound is prepared either in physiologic saline containing 0.05% Tween 80 or distilled water containing 5% dextrose and is administered intraperitoneally on the indicated schedule relative to tumor implant. Doses are on a mg/kg basis according to individual animals' body weights. The day of death for each animal is recorded, the median identified for each group and the percent increase in life span (% ILS) is calculated from the ratio of survival time of treated to control groups. The criterion for activity is % ILS greater than or equal to 20%.
-
- The two routine indicator cell lines used are 1.) D98, a twice- cloned derivative of Detroit 98s from American Type Culture Collection (ATCC) strain CCL 18.1, from human sternal bone marrow and 2.) L, a cloned derivative from NCTC 929 (ATCC CCL1,) a C₃H/An from mouse connective tissue. Inoculation densities for L and D98 cells are 1.0 and 1.5 x 10⁵ cells, per cm², respectively. Control cells are grown in Eagle's MEM(Earle's salts, 10% horse serum, 100 units/ml potassium penicillin G and 100 µg/ml streptomycin) and should show at least two doublings during the test period. Test plates are seeded with similar quantities of cells and are grown in the same medium with test compounds added. Total "cell counts are made with an electronic cell counter after 70-76 hours of incubation with one medium change at 20-26 hours. Dose response curves are prepared by plotting percent of control vs. concentration of compound.
-
- From the foregoing data, it can, therefore, be concluded that the compounds of the present invention are active in both the lymphocytic leukemia P388/0 and the cell culture cytotoxicity tests and, therefore, that the compounds have anti-neoplastic activity and are able to inhibit neoplastic growth.
- As mentioned previously, it may be necessary, in the treatment of prophylaxis of a neoplastic growth, to administer to the patient an antidote or rescue agent. Demonstration of the efficacy in this regard of calcium leucovorin, hypoxanthine and AICAR was carried out on mouse L cells using the compounds of Examples 1 and 3. The results are as follows.
- From the foregoing data, it can, therefore, be concluded that each of calcium leucovorin and hypoxanthine are able to reverse the cytotoxicity of the compound of Example 1 and that each of calcium leucovorin, hypoxanthine and AICAR are able to reverse the cytotoxicity of the compound of Example 3.
- Frozen broth cultures are diluted to a predetermined titre of 100 color changing units for the test. Organisms are grown in Difco PPLO broth base with added serum, glucose, phenol red, ampicillin, thallium acetate and yeast extract. Incubation is at 32oC. Incubation with varying levels of the test compound in the above medium are performed in well plates and an amount of Spiroplasma citri cells is added that will produce a color change by a fixed time of growth. The lowest concentration of compound that fails to produce a color change or gives a two day delay in color change indicates minimum inhibitory concentration.
-
- From the foregoing data, it can, therefore, be concluded that the compounds of Examples 1 to 3 are active against the mycoplasma, Spiroplasma citri.
Claims (11)
- (1) A process for the preparation of a compound of formula (I):
- (2) A process for the preparation of a compound of formula (I) according to Claim 1, wherein NR¹ is NH or NCHO.
- (3) A process for the preparation of a compound of formula (I) according to Claim 1, wherein R⁴ and NR⁵R⁶ are both NH₂.
- (4) A process for the preparation of a compound of formula (I) according to Claim 1, wherein R², R³, R⁷, R⁸, R⁹ and R¹⁰ are all hydrogen.
- (5) A process for the preparation of a compound of formula (I) according to Claim 1, wherein n is 3 or 4.
- (6) A process for the preparation of a compound of formula (I) according to Claim 1, wherein m is 0.
- (7) A process according to Claim 1 for preparing a compound selected from the group comprising: N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propylamino]-benzoyl]-L-glutamic acid,
N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-N-methyl-propylamino]benzoyl]-L-glutamic acid,
N-[4-[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)-N-formyl-propylamino]benzoyl]-L-glutamic acid,
N-[4-[2-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethylamino]-benzoyl]-L-glutamic acid,
N-[4-[2-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)ethyl-N-methylamino]benzoyl]-L-glutamic acid, or salts thereof, - (8) A pharmaceutical formulation, which comprises a compound according to Claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.
- (9) A compound of formula (I) or a salt thereof, as defined in Claim 1, for use in the manufacture of a medicament.
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GB868625019A GB8625019D0 (en) | 1986-10-18 | 1986-10-18 | Compounds |
GB8625019 | 1986-10-18 |
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EP0268377A3 EP0268377A3 (en) | 1988-12-28 |
EP0268377B1 EP0268377B1 (en) | 1992-03-18 |
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US (1) | US4880812A (en) |
EP (1) | EP0268377B1 (en) |
JP (1) | JPS63126868A (en) |
AT (1) | ATE73780T1 (en) |
AU (1) | AU598307B2 (en) |
CA (1) | CA1300807C (en) |
DE (1) | DE3777544D1 (en) |
ES (1) | ES2030735T3 (en) |
GB (1) | GB8625019D0 (en) |
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Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0324270A1 (en) * | 1987-12-28 | 1989-07-19 | Nippon Mining Company Limited | Biologically active peptides |
US4880812A (en) * | 1986-10-18 | 1989-11-14 | Burroughs Wellcome Co. | Pyrimidine compounds |
EP0341837A2 (en) * | 1988-04-15 | 1989-11-15 | The Wellcome Foundation Limited | Glutamic acid derivatives |
US4921836A (en) * | 1989-05-17 | 1990-05-01 | Burroughs Wellcome Co. | Substituted glutamic acids |
US4940713A (en) * | 1988-04-15 | 1990-07-10 | Burroughs Wellcome Co. | Substituted glutamic acids |
US4971973A (en) * | 1989-05-17 | 1990-11-20 | Burroughs Wellcome Co. | Glutamic acid derivatives |
WO1990014340A1 (en) * | 1989-05-17 | 1990-11-29 | The Wellcome Foundation Limited | Glutamic acid derivatives |
EP0402903A1 (en) * | 1989-06-14 | 1990-12-19 | Takeda Chemical Industries, Ltd. | Production of pyrrolopyrimidines and intermediates therefor |
US5019577A (en) * | 1988-04-15 | 1991-05-28 | Burroughs Welcome Co. | Novel compounds and use |
EP0492316A1 (en) * | 1990-12-14 | 1992-07-01 | Takeda Chemical Industries, Ltd. | Condensed heterocyclic oligoglutamate derivatives, their production and pharmaceutical compositions containing them |
US5444061A (en) * | 1991-03-19 | 1995-08-22 | British Technology Group Limited | Anti-cancer compounds |
US5561133A (en) * | 1992-03-18 | 1996-10-01 | British Technology Group Limited | Thymidylate synthase inhibiting quinazolinones |
US5594139A (en) * | 1993-01-29 | 1997-01-14 | Agouron Pharmaceuticals, Inc. | Processes for preparing antiproliferative garft-inhibiting compounds |
US5726312A (en) * | 1992-12-16 | 1998-03-10 | Agouron Pharmaceuticals, Inc. | Methods for preparing antiproliferative 5-substituted pyrimidone compounds |
US5831100A (en) * | 1995-06-07 | 1998-11-03 | Agouron Pharmaceuticals, Inc. | Syntheses of optically pure compounds useful as GARFT inhibitors and their intermediates |
US5945427A (en) * | 1995-06-07 | 1999-08-31 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
WO2003074083A1 (en) * | 2002-03-04 | 2003-09-12 | Pfizer Inc. | Combination therapies for treating methylthioadenosine phosphorylase deficient cells |
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IT1229203B (en) * | 1989-03-22 | 1991-07-25 | Bioresearch Spa | USE OF 5 METHYLTHETRAHYDROPHOLIC ACID, 5 FORMYLTHETRAHYDROPHOLIC ACID AND THEIR PHARMACEUTICALLY ACCEPTABLE SALTS FOR THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS IN THE FORM OF CONTROLLED RELEASE ACTIVE IN THE THERAPY OF MENTAL AND ORGANIC DISORDERS. |
US5013738A (en) * | 1990-04-18 | 1991-05-07 | The Trustees Of Princeton University | L-glutamic acid derivatives |
AU2001282520A1 (en) * | 2000-08-29 | 2002-03-13 | Takeda Chemical Industries Ltd. | Grk inhibitor |
US20040170705A1 (en) * | 2001-03-08 | 2004-09-02 | Dusan Miljkovic | Compositions and methods for non-insulin glucose uptake |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8607683D0 (en) * | 1986-03-27 | 1986-04-30 | Ici Plc | Anti-tumor agents |
NZ219971A (en) * | 1986-06-06 | 1989-08-29 | Univ Princeton | Tetrahydropyrido (2,3-d pyrimidine derivatives and pharmaceutical compositions |
IE60038B1 (en) * | 1986-06-30 | 1994-05-18 | Univ Princeton | 4(3H)-oxo-5, 6, 7, 8-tetrahydropyrido-[2, 3-d]pyrimidine derivatives |
GB8625019D0 (en) * | 1986-10-18 | 1986-11-19 | Wellcome Found | Compounds |
-
1986
- 1986-10-18 GB GB868625019A patent/GB8625019D0/en active Pending
-
1987
- 1987-10-16 JP JP62261498A patent/JPS63126868A/en active Pending
- 1987-10-16 US US07/109,225 patent/US4880812A/en not_active Expired - Fee Related
- 1987-10-16 CA CA000549535A patent/CA1300807C/en not_active Expired - Fee Related
- 1987-10-16 AU AU79871/87A patent/AU598307B2/en not_active Ceased
- 1987-10-16 AT AT87309165T patent/ATE73780T1/en active
- 1987-10-16 ES ES198787309165T patent/ES2030735T3/en not_active Expired - Lifetime
- 1987-10-16 ZA ZA877801A patent/ZA877801B/en unknown
- 1987-10-16 DE DE8787309165T patent/DE3777544D1/en not_active Expired - Fee Related
- 1987-10-16 EP EP87309165A patent/EP0268377B1/en not_active Expired - Lifetime
-
1992
- 1992-04-06 GR GR920400628T patent/GR3004273T3/el unknown
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Journal of Medicinal Chemistry, Vol. 6, November 1963, pages 664-669, B.R. BAKER et al.: "Analogs of tetrahydrofolic acid. IX. Synthesis of N-(1-(2-amino-4-hydroxy-6-phenyl-5-pyrimidyl)-3-propyl)-p-aminobenzoyl-L-glutamic acid, a 'nonclassical' inhibitor of some folic cofactor area enzymes", whole article. * |
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US4880812A (en) * | 1986-10-18 | 1989-11-14 | Burroughs Wellcome Co. | Pyrimidine compounds |
EP0324270A1 (en) * | 1987-12-28 | 1989-07-19 | Nippon Mining Company Limited | Biologically active peptides |
US5019577A (en) * | 1988-04-15 | 1991-05-28 | Burroughs Welcome Co. | Novel compounds and use |
EP0341837A2 (en) * | 1988-04-15 | 1989-11-15 | The Wellcome Foundation Limited | Glutamic acid derivatives |
US4940713A (en) * | 1988-04-15 | 1990-07-10 | Burroughs Wellcome Co. | Substituted glutamic acids |
EP0341837A3 (en) * | 1988-04-15 | 1990-08-16 | The Wellcome Foundation Limited | Glutamic acid derivatives |
US4921836A (en) * | 1989-05-17 | 1990-05-01 | Burroughs Wellcome Co. | Substituted glutamic acids |
US4971973A (en) * | 1989-05-17 | 1990-11-20 | Burroughs Wellcome Co. | Glutamic acid derivatives |
WO1990014340A1 (en) * | 1989-05-17 | 1990-11-29 | The Wellcome Foundation Limited | Glutamic acid derivatives |
EP0409382A1 (en) * | 1989-05-17 | 1991-01-23 | The Wellcome Foundation Limited | Glutamic acid derivatives |
US5496946A (en) * | 1989-06-14 | 1996-03-05 | Takeda Chemical Industries, Ltd. | Production of pyrrolopyrimidines and intermediates therefor |
US5349064A (en) * | 1989-06-14 | 1994-09-20 | Takeda Chemical Industries, Ltd. | Production of pyrrolopyrimidines |
EP0402903A1 (en) * | 1989-06-14 | 1990-12-19 | Takeda Chemical Industries, Ltd. | Production of pyrrolopyrimidines and intermediates therefor |
CN1051306C (en) * | 1989-06-14 | 2000-04-12 | 武田药品工业株式会社 | Production of pyrrolopyrimidines and intermadiates therefor |
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US5268362A (en) * | 1990-12-14 | 1993-12-07 | Takeda Chemical Industries, Ltd. | Condensed heterocyclic compounds, their production and use |
US5459129A (en) * | 1990-12-14 | 1995-10-17 | Takeda Chemical Industries, Ltd. | Condensed heterocyclic compounds, their production and use |
US5444061A (en) * | 1991-03-19 | 1995-08-22 | British Technology Group Limited | Anti-cancer compounds |
US5561133A (en) * | 1992-03-18 | 1996-10-01 | British Technology Group Limited | Thymidylate synthase inhibiting quinazolinones |
US6323210B2 (en) | 1992-12-16 | 2001-11-27 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
US6207670B1 (en) | 1992-12-16 | 2001-03-27 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
US5739141A (en) * | 1992-12-16 | 1998-04-14 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
US5726312A (en) * | 1992-12-16 | 1998-03-10 | Agouron Pharmaceuticals, Inc. | Methods for preparing antiproliferative 5-substituted pyrimidone compounds |
US5641774A (en) * | 1993-01-29 | 1997-06-24 | Agouron Pharmaceuticals, Inc. | Compounds useful as antiproliferative agents and garft inhibitors |
US5641771A (en) * | 1993-01-29 | 1997-06-24 | Agouron Pharmaceuticals, Inc. | Compounds useful as antiproliferative agents and garft inhibitors |
US5723607A (en) * | 1993-01-29 | 1998-03-03 | Agouron Pharmaceticals, Inc. | Compounds useful as antiproliferative agents and GARFT inhibitors |
US5639747A (en) * | 1993-01-29 | 1997-06-17 | Agouron Pharmaceuticals, Inc. | Compounds useful as antiproliferative agents and garft inhibitors |
US5639749A (en) * | 1993-01-29 | 1997-06-17 | Agouron Pharmaceuticals, Inc. | Compounds useful as antiproliferative agents and garft inhibitors |
US5625061A (en) * | 1993-01-29 | 1997-04-29 | Agouron Pharmaceuticals, Inc. | Compounds useful in preparing antiproliferative agents and garft inhibitors |
US5610319A (en) * | 1993-01-29 | 1997-03-11 | Agouron Pharmaceuticals, Inc. | Compound useful as antiproliferative agents and GARFT inhibitors |
US5594139A (en) * | 1993-01-29 | 1997-01-14 | Agouron Pharmaceuticals, Inc. | Processes for preparing antiproliferative garft-inhibiting compounds |
US5831100A (en) * | 1995-06-07 | 1998-11-03 | Agouron Pharmaceuticals, Inc. | Syntheses of optically pure compounds useful as GARFT inhibitors and their intermediates |
US5945427A (en) * | 1995-06-07 | 1999-08-31 | Agouron Pharmaceuticals, Inc. | Antiproliferative substituted 5-thiapyrimidinone and 5-selenopyrimidinone compounds |
US5981748A (en) * | 1995-06-07 | 1999-11-09 | Agouron Pharmaceuticals, Inc. | Synthesis of optically pure compounds useful as GARFT inhibitors and their intermediates |
WO2003074083A1 (en) * | 2002-03-04 | 2003-09-12 | Pfizer Inc. | Combination therapies for treating methylthioadenosine phosphorylase deficient cells |
Also Published As
Publication number | Publication date |
---|---|
DE3777544D1 (en) | 1992-04-23 |
US4880812A (en) | 1989-11-14 |
ZA877801B (en) | 1989-05-30 |
EP0268377A3 (en) | 1988-12-28 |
AU598307B2 (en) | 1990-06-21 |
ATE73780T1 (en) | 1992-04-15 |
JPS63126868A (en) | 1988-05-30 |
CA1300807C (en) | 1992-05-12 |
EP0268377B1 (en) | 1992-03-18 |
GB8625019D0 (en) | 1986-11-19 |
AU7987187A (en) | 1988-04-21 |
ES2030735T3 (en) | 1992-11-16 |
GR3004273T3 (en) | 1993-03-31 |
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